Novel compositions and methods for the treatment of immune related diseases

ABSTRACT

The present invention relates to compositions and methods of using those compositions for the diagnosis and treatment of immune related diseases.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional application No. 61/123,530, filed Apr. 9, 2008, and to U.S.provisional application No. 61/194,271, filed Sep. 26, 2008, thecontents of which are incorporated in their entirety herein byreference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful for thediagnosis and treatment of immune related diseases.

BACKGROUND OF THE INVENTION

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases,non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response. Another subcategory of helper T cells are thefollicular helper T cells (T_(Fh)) (for review, see Vineusa et al., Nat.Rev. Immunol. 5: 853-865 (2005)). Detectable by their characteristicexpression of CXC-chemokine receptor 5 (Schaerli et al., J. Exp. Med.192: 1553-62 (2000)), these cells have been found to produce IL-10 andpossibly IL-21. T_(Fh) cells provide assistance to germinal-center Bcells, particularly aiding the survival and propagation of B cells andpotently inducing antibody production during coculture with B cells.They have also been implicated in tolerogenesis.

Regulatory T cells (T_(reg)) are a subset of helper T cells that play acritical role in inhibition of self-reactive immune responses and areoften found in sites of chronic inflammation such as in tumor tissue(Wang, H. Y. & Wang, R. F., Curr Opin Immunol 19, 217-23 (2007)).T_(regs) are defined phenotypically by high cell surface expression ofCD25, CLTA4, GITR, and neuropilin-1 (Read, S., Malmstrom, V. & Powrie,F., J Exp Med 192, 295-302 (2000); Sakaguchi, S., et al., J Immunol 155,1151-64 (1995); Takahashi, T. et al., J Exp Med 192, 303-10 (2000);McHugh, R. S. et al., Immunity 16, 311-23 (2002); Bruder, D. et al., EurJ Immunol 34, 623-30 (2004)), and are under the control of thetranscription factor FOXP3 (Hori, S., Nomura, T. & Sakaguchi, S.,Science 299, 1057-61 (2003)). T_(regs) perform their suppressivefunction on activated T cells through contact-dependent mechanisms andcytokine production (Fehervari, Z. & Sakaguchi, Curr Opin Immunol 16,203-8 (2004)). T_(regs) also modulate immune responses by directinteraction with ligands on dendritic cells (DC), such as CTLA4interaction with B7 molecules on DC that elicits the induction ofindoleamine 2,3-dioxygenase (IDO) (Fallarino, F. et al., Nat Immunol 4,1206-12 (2003)), and CD40L ligation (Serra, P. et al., Immunity 19,877-89 (2003)). DCs are professional antigen-presenting cells capable ofinducing immunity or tolerance against self or non-self antigens.DC-expanded T_(regs) suppress alloreactivity responses in vitro(Yamazaki, S. et al., Proc Natl Acad Sci USA 103, 2758-63 (2006); Ahn,J. S., Krishnadas, D. K. & Agrawal, Int Immunol 19, 227-37 (2007)), andwhen adoptively transferred, appropriate T_(regs) inhibited diabetes inNOD.scid mice (Tarbell, K. V. et al., J Exp Med 199, 1467-77 (2004)) orexperimentally induced asthma (Lewkowich, I. P. et al. J Exp Med 202,1549-61 (2005)). Specific interactions of ligands on DC with T_(regs)can also abrogate their suppressive function, such as engagement of GITRin mice (Shimizu, J., et al., Nat Immunol 3, 135-42 (2002)), suggestingDC may have a pluralistic role in modulating T_(reg) function.

The molecules CTLA4 and GITR are representative of ligands definedwithin the CD28-B7 and TNF-superfamilies of co-stimulatory/-inhibitorymolecules, respectively (Greenwald, R. J., et al., Annu Rev Immunol 23,515-48 (2005)). These molecules are high on T_(regs) but are alsotypically upregulated on activated T cells. In order to search for newco-stimulatory molecules expressed in T_(reg) cells searches wereperformed to identify genes specifically expressed in T cells (Abbas, A.R. et al., Genes Immun 6, 319-31 (2005)) that had both Ig domains andimmunoreceptor tyrosine-based activation or inhibition (ITAM/ITIM)motifs. Through the intersection of these two genome-wide bioinformaticssearch strategies a novel cell surface-bound protein with the proteinencoding an IgV domain, a transmembrane domain, and two putativeimmunoreceptor tyrosine inhibitory motifs was identified (see US patentpublication no. US20040121370, incorporated herein by reference). Theprotein designated TIGIT (for T-Cell-Ig and ITIM domain) was shown to beexpressed on T cells—particularly T_(reg) and memory cell subsets—aswell as NK cells. There is a need for new therapeutics and methods oftreatment to address immune disorders, particularly autoimmunedisorders. Herein, Applicants identify TIGIT binding partners andprovide new compositions, detection methods, and methods of treatmentfor immune disorders modulated by TIGIT interaction with those bindingpartners and the elucidated TIGIT effects on T cell maturation andactivity.

SUMMARY OF THE INVENTION

The present invention concerns compositions and methods useful for thediagnosis and treatment of immune related disease in mammals, includinghumans. The present invention is based on the identification of proteinsinvolved in the negative regulation of proliferation and function ofcertain types of immune cells. Immune related diseases can be treated bysuppressing or enhancing the immune response. Molecules that enhance theimmune response stimulate or potentiate the immune response to anantigen. Molecules which stimulate the immune response can be usedtherapeutically where enhancement of the immune response would bebeneficial. Alternatively, molecules that suppress the immune responseattenuate or reduce the immune response to an antigen (e.g.,neutralizing antibodies) can be used therapeutically where attenuationof the immune response would be beneficial (e.g., inflammation). Herein,Applicants demonstrate that TIGIT (for “T-Cell-Ig and ITIM domain”)protein specifically binds to poliovirus receptor (PVR, also known asCD155) and several other members of a newly elucidated protein family,and that this TIGIT-PVR interaction negatively regulates T cellactivation and proliferation. Accordingly, TIGIT polypeptides, agoniststhereof, and antagonists thereof, as well as PVR polypeptides, agoniststhereof and antagonists thereof are useful to prepare medicines andmedicaments for the treatment of immune-related and inflammatorydiseases. The invention also provides methods of treating immune-relatedand inflammatory diseases and methods and compositions for detecting andassessing the status of immune-related and inflammatory diseases.

In one embodiment, the invention provides an isolated polypeptidecomprising an amino acid sequence comprising one or more of thefollowing amino acids: an alanine at amino acid position correspondingto amino acid position 67 of human TIGIT, a glycine at an amino acidposition corresponding to amino acid position 74 of human TIGIT, aproline at an amino acid position corresponding to amino acid position114 of human TIGIT, and a glycine at an amino acid positioncorresponding to amino acid position 116 of human TIGIT. In one aspect,the polypeptide is not PVR, PVRL1, PVRL2, PVRL3, PVRL4, TIGIT, CD96, orCD226. In another aspect, the polypeptide further comprises one or moreof: an amino acid selected from valine, isoleucine, and leucine at anamino acid position corresponding to amino acid position 54 of humanTIGIT, an amino acid selected from serine and threonine at an amino acidposition corresponding to amino acid position 55 of human TIGIT, aglutamine at an amino acid position corresponding to amino acid position56 of human TIGIT, a threonine at an amino acid position correspondingto amino acid position 112 of human TIGIT, and an amino acid selectedfrom phenylalanine and tyrosine at an amino acid position correspondingto amino acid position 113 of human TIGIT. In another aspect, thepolypeptide further comprises one or more structural submotifs selectedfrom the following:

-   -   a. an amino acid selected from valine and isoleucine at amino        acid position 54-an amino acid selected from serine and        threonine at amino acid position 55-a glutamine at amino acid        position 56;    -   b. an alanine at position 67-any amino acid at each of amino        acid positions 68-73-a glycine at amino acid position 74; and    -   c. a threonine at amino acid position 112-an amino acid selected        from phenylalanine and tyrosine at amino acid position 113-a        proline at amino acid position 114-any amino acid at amino acid        position 115-a glycine at amino acid position 116,        wherein the numbering of the amino acid positions corresponds to        the amino acid positions of human TIGIT, although the absolute        numbering of the amino acids in the polypeptide may differ.

In another embodiment, the invention provides a method of determiningwhether a test polypeptide is a member of the TLP family of polypeptidescomprising aligning the amino acid sequence of the test polypeptide withan amino acid sequence of one or more members of the TLP family ofpolypeptides and assessing the presence or absence in the testpolypeptide amino acid sequence of one or more of an alanine at aminoacid position corresponding to amino acid position 67 of human TIGIT, aglycine at an amino acid position corresponding to amino acid position74 of human TIGIT, a proline at an amino acid position corresponding toamino acid position 114 of human TIGIT, and a glycine at an amino acidposition corresponding to amino acid position 116 of human TIGIT. Inanother embodiment, the invention provides a method for identifying oneor more members of the TLP protein family by identifying proteins in oneor more sequence databases whose amino acid sequences comprise at leastone amino acid selected from an alanine at amino acid positioncorresponding to amino acid position 67 of human TIGIT, a glycine at anamino acid position corresponding to amino acid position 74 of humanTIGIT, a proline at an amino acid position corresponding to amino acidposition 114 of human TIGIT, and a glycine at an amino acid positioncorresponding to amino acid position 116 of human TIGIT.

In another embodiment, the invention provides an isolated agent thatspecifically interacts with one or more conserved or substantiallyconserved regions of TLP family members. In one aspect, the agent is anantagonist of the expression and/or activity of a TLP family member. Inanother aspect, the antagonist is selected from a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.In another aspect, the agent is an agonist of the expression and/oractivity of a TLP family member. In another aspect, the agonist isselected from an agonizing antibody or antigen-binding fragment thereof,an agonizing peptide, and a small molecule or protein that activatesTIGIT binding to PVR and/or TIGIT intracellular signaling mediated byPVR. In another embodiment, the invention provides a method ofidentifying or detecting one or more TLP family members by contacting aputative TLP family member polypeptide with at least one of the aboveagents and determining the binding of the at least one agent to theputative TLP family member.

In another embodiment, the invention provides a method of determiningwhether a test immune cell is an activated or normal T_(reg), memory Tcell, NK cell, or T_(Fh) cell, comprising assessing the level ofexpression of TIGIT in the test immune cell and comparing it to thelevel of expression of TIGIT in a known activated or normal T_(reg),memory T cell, NK cell, or TFh cell, or by comparing the level ofexpression of TIGIT in the test immune cell to known standard TIGITexpression value(s). In another embodiment, the invention provides amethod for modulating immune system function and/or activity comprisingmodulating the binding of TIGIT to one or more of PVR, PVRL3, and PVRL2.

In another embodiment, the invention provides an anti-TIGIT antibody ora fragment thereof comprising at least one HVR comprising an amino acidsequence selected from the amino acid sequences set forth in SEQ ID NOs:23-28. In another embodiment, the invention provides an anti-TIGITantibody or a fragment thereof comprising at least one HVR comprising anamino acid sequence selected from the amino acid sequences set forth inSEQ ID NOs: 31-36. In another embodiment, the invention provides ananti-TIGIT antibody or a fragment thereof wherein the antibody lightchain comprises the amino acid sequence set forth in SEQ ID NO: 21. Inanother embodiment, the invention provides an anti-TIGIT antibody or afragment thereof wherein the antibody light chain comprises the aminoacid sequence set forth in SEQ ID NO: 29. In another embodiment, theinvention provides an anti-TIGIT antibody or a fragment thereof whereinthe antibody heavy chain comprises the amino acid sequence set forth inSEQ ID NO: 22 or a portion thereof. In another embodiment, the inventionprovides an anti-TIGIT antibody or a fragment thereof wherein theantibody heavy chain comprises the amino acid sequence set forth in SEQID NO: 30 or a portion thereof. In another embodiment, the inventionprovides an anti-TIGIT antibody or a fragment thereof wherein theantibody light chain comprises the amino acid sequence set forth in SEQID NO: 21 or a portion thereof and the antibody heavy chain comprisesthe amino acid sequence set forth in SEQ ID NO: 22 or a portion thereof.In another embodiment, the invention provides an anti-TIGIT antibody ora fragment thereof wherein the antibody light chain comprises the aminoacid sequence set forth in SEQ ID NO: 29 or a portion thereof and theantibody heavy chain comprises the amino acid sequence set forth in SEQID NO: 30 or a portion thereof. In another embodiment, the inventionprovides an anti-TIGIT antibody or a fragment thereof wherein theantibody light chain is encoded by the nucleotide sequence of SEQ ID NO:50 or a portion thereof. In another embodiment, the invention providesan anti-TIGIT antibody or a fragment thereof wherein the antibody heavychain is encoded by the nucleotide sequence of SEQ ID NO: 51 or aportion thereof. In one aspect, an antibody or antigen-binding fragmentthereof of the invention is selected from a humanized antibody, achimeric antibody, a bispecific antibody, a heteroconjugate antibody,and an immunotoxin.

In another aspect, the at least one HVR of the invention is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an HVRset forth in any of SEQ ID NOs: 23-28. In another aspect, the at leastone HVR of the invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an HVR set forth in any of SEQ ID NOs:31-36. In another aspect, the light chain of an antibody orantigen-binding fragment of the invention comprises an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence set forth in SEQ ID NO: 21. Inanother aspect, the light chain of an antibody or antigen-bindingfragment of the invention comprises an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence set forth in SEQ ID NO: 29. In another aspect, the heavychain of an antibody or antigen-binding fragment of the inventioncomprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence set forth inSEQ ID NO: 22. In another aspect, the heavy chain of an antibody orantigen-binding fragment of the invention comprises an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence set forth in SEQ ID NO: 30. Inanother aspect, an antibody or antigen-binding fragment of the inventioncomprises a light chain comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence set forth in SEQ ID NO: 21 and a heavy chain comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:22. In another aspect, an antibody or antigen-binding fragment of theinvention comprises a light chain comprising an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence set forth in SEQ ID NO: 29 and a heavy chaincomprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence set forth inSEQ ID NO: 30.

In another embodiment, the invention provides a method of modulating aCD226-PVR interaction and/or a CD96-PVR interaction comprisingadministering at least one of TIGIT, an agonist of TIGIT expressionand/or activity, or an antagonist of TIGIT expression and/or activity invivo or in vitro. In one aspect, TIGIT or an agonist of TIGIT expressionand/or activity is administered and the CD226-PVR interaction and/or theCD96-PVR interaction is inhibited or blocked. In another aspect, anantagonist of TIGIT expression and/or activity is administered and theCD226-PVR interaction and/or the CD96-PVR interaction is stimulated.

In another embodiment, the invention provides a method of modulatingimmune cell function and/or activity by modulating TIGIT and/or PVRexpression and/or activity, or by modulating the intracellular signalingmediated by TIGIT binding to PVR. In one aspect, the modulating isdecreasing or inhibiting proliferation of one or more immune cells orproinflammatory cytokine release by one or more immune cells by treatingthe cells in vitro or in vivo with TIGIT, an agonist of TIGIT expressionand/or activity, an agonist of PVR expression and/or activity, or bystimulating intracellular signaling mediated by TIGIT binding to PVR. Inanother aspect, the modulating is increasing or stimulatingproliferation of one or more immune cells or proinflammatory cytokinerelease by one or more immune cells by treating the cells in vitro or invivo with an antagonist of TIGIT expression and/or activity, anantagonist of PVR expression and/or activity, or by inhibitingintracellular signaling mediated by TIGIT binding to PVR.

In another embodiment, the invention provides a method of inhibiting animmune response by administering in vitro or in vivo TIGIT, an agonistof TIGIT expression and/or activity, an agonist of PVR expression and/oractivity, or by stimulating intracellular signaling mediated by TIGITbinding to PVR. In another embodiment, the invention provides a methodof increasing or stimulating an immune response by administering invitro or in vivo an antagonist of TIGIT expression and/or activity, anantagonist of PVR expression and/or activity, or by inhibitingintracellular signaling mediated by TIGIT binding to PVR.

In another embodiment, the invention provides a method of modulating thetype and/or amount of cytokine production from an immune cell bymodulating TIGIT or PVR expression and/or activity in vitro or in vivo.In one aspect, proinflammatory cytokine production is stimulated and/orincreased by administration of an antagonist of TIGIT expression and/oractivity, an antagonist of PVR expression and/or activity, or byinhibiting intracellular signaling mediated by TIGIT binding to PVR. Inanother aspect, proinflammatory cytokine production is inhibited byadministration of an agonist of TIGIT expression and/or activity, anagonist of PVR expression and/or activity, or by stimulatingintracellular signaling mediated by TIGIT binding to PVR.

In another embodiment, the invention provides a method of stimulatingERK phosphorylation and/or intracellular signaling through the ERKpathway in one or more immune cells comprising treating the one or moreimmune cells with TIGIT, an agonist of TIGIT expression and/or activity,or an agonist of PVR expression and/or activity.

In another embodiment, the invention provides a method of diagnosing animmune-related disease relating to aberrant immune cell response in asubject comprising assessing the expression and/or activity of TIGIT ina sample from the subject and comparing the expression and/or activityof TIGIT to a reference amount of TIGIT expression and/or activity orthe amount of TIGIT expression and/or activity in a sample from a normalsubject. In one aspect, the immune-related disease is selected frompsoriasis, arthritis, inflammatory bowel disease or cancer. In anotheraspect, the cancer is breast cancer. In another embodiment, theinvention provides a method of assessing the severity of animmune-related disease relating to aberrant immune cell response in asubject comprising assessing the expression and/or activity of TIGIT ina sample from the subject and comparing the expression and/or activityof TIGIT to a reference amount of TIGIT expression and/or activity orthe amount of TIGIT expression and/or activity in a sample from a normalsubject. In one aspect, the immune-related disease is selected frompsoriasis, arthritis, inflammatory bowel disease or cancer. In anotheraspect, the cancer is breast cancer. In another embodiment, theinvention provides a method of preventing an immune-related diseaserelating to aberrant immune cell response in a subject comprisingmodulating the expression and/or activity of TIGIT in the subject. Inone aspect, the immune-related disease is selected from psoriasis,arthritis, inflammatory bowel disease or cancer. In another aspect, thecancer is breast cancer. In another embodiment, the invention provides amethod of treating or lessening the severity of an immune-relateddisease relating to aberrant immune cell response in a subjectcomprising modulating the expression and/or activity of TIGIT in thesubject. In one aspect, the immune-related disease is selected frompsoriasis, arthritis, inflammatory bowel disease or cancer. In anotheraspect, the cancer is breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of human, mouse, rhesus and dog TIGITprotein sequences. Shading indicates positions containing identicalamino acids in three or four species. The signal sequence is indicatedby a dashed line, the immunoglobulin V-set domain is indicated by adouble line, N-glycosylation sites are indicated by a thin line abovethe requisite position, the transmembrane domain is indicated by a thickline, and the putative extended ITIM motif is indicated by a doubledashed line. Human TIGIT shares 88%, 67%, and 58% identity with rhesus,dog and mouse sequences, respectively.

FIGS. 2A and 2B depict an alignment of protein sequences of IgV domainsof the indicated PVR family proteins. Side chains that share similarityacross sequences are marked according to property. V-frame fingerprintresidues (black circle) and PVR-related fingerprint residues (thick lineboxed residues) are indicated. For comparative purposes, six IgV domainsequences (set forth under the horizontal line) from non-PVR familymembers are also aligned.

FIG. 3 depicts the results of biosensor analyses to assess the abilityof TIGIT-Fc (light grey line) or a control-Fc protein (black line) tobind to various proteins, as described in Example 2. The numbers 1-8represent, respectively, ESAM, OTOR, TEK, TNFRSF10C, IGFBP4, PVR, IL-19,and TEK.

FIG. 4A depicts the results of biosensor assays to assess the binding ofvarious Fc fusion proteins to immobilized TIGIT-Fc, as described inExample 2. FIGS. 4B-1 to 4B-6 depict the results of FACS analyses toassess the binding of biotinylated Fc-fusion proteins toreceptor-expressing CHO stable transfectants, as described in Example 2.

FIGS. 5A and 5B depict the results of one representative radioligandbinding assay to determine the Kd for binding between TIGIT-Fc andPVR-expressing CHO cells, as described in Example 2.

FIG. 6 shows graphs depicting the results of competition binding studiesamong TIGIT, PVR, CD226 and CD96, as described in Example 2.

FIG. 7 shows the results of experiments assessing the ability of ananti-PVR antibody to block PVR binding to TIGIT or CD226, as describedin Example 2. FIG. 7A depicts the binding of biotinylated PVR-Fc to CHOtransfectants expressing CD226 or TIGIT in the presence (dotted line) orabsence (solid line) of a 10-fold molar excess of antibody D171. Theresults from a matched isotype control antibody are indicated by theshaded area. FIG. 7B depicts the binding of PVR-Fc (top line) or buffer(bottom line) to biosensors loaded with CD226-Fc or TIGIT-Fc. The middleline indicates PVR-Fc binding to biosensor preloaded CD226-Fc orTIGIT-Fc that had been blocked with antibody D171 prior to exposure toPVR-Fc.

FIG. 8A depicts TIGIT expression data (left panel) or CD226 expressiondata (right panel) in a variety of immune cell types, as described inExample 2(A). FIG. 8B depicts RT-PCR analyses of TIGIT and ICOS mRNAexpression in tonsillar T_(fh) cells, as described in Example 2(A).

FIGS. 9A-B depict the results of experiments testing the ability ofanti-TIGIT antibody 10A7 to bind to TIGIT at the surface of cells, asdescribed in Example 3. FIG. 9A shows the binding of anti-TIGIT antibody10A7 to stable 293-TIGIT cell lines (solid line) and the abrogation ofthat binding in the presence of PVR-Fc (dashed line). The grey regionrepresents the binding of an isotype-matched control antibody. FIG. 9Bshows the results of FACS analyses demonstrating that TIGIT co-expresseswith FoxP3 in GITR⁺ CD4 T-cells. The data shown is representative of twoindependent experiments.

FIGS. 10A-F depict the results of experiments assessing TIGIT expressioneither by mRNA analysis or by binding studies at the cell surface, asdescribed in Example 3. FIGS. 10A-1 to 10A-2 depict the results of flowcytometric experiments to determine the expression of TIGIT and CD226 onresting or activated (for one or two days) CD4⁺CD45RA⁺ (left panel) orCD4⁺CD45RO⁺ T cells (right panel), as described in Example 2(A). FIG.10B shows a bar graph indicating the fold-change in TIGIT mRNA indifferent types of immune cells sorted directly ex vivo from PBMC, ascompared to the TIGIT mRNA levels in naïve CD4⁺CD45RA⁺ cells. FIG. 10Cshows bar graphs indicating the -fold increase in TIGIT mRNA levels onsorted CD4⁺CD45RO⁺, CD4⁺CD45RA⁺ and CD4⁺CD25^(hi) T_(reg) cellsactivated with anti-CD3 and anti-CD28 for 1 or 2 days or sorted CD56⁺ NKcells activated with IL-2 for one day, as compared to unstimulatedcells. The FACS plots shown are from one representative experiment andthe RT-PCR values are an average of three donors. FIG. 10D shows theresults of FACS assays showing that CD25⁻ human PBMC cells lack TIGITexpression. FIG. 10E depicts the results of FACS experiments assessingthe cell surface expression of TIGIT on human PBMC cells expressing lowor high amounts of CD25 and shows that expression of TIGIT correlateswith expression of FOXP3. FIG. 10F depicts the results of FACSexperiments assessing TIGIT expression in sorted CD4⁺CD25^(hi) T cellsactivated with anti-CD3 and anti-CD28 for 24 hours (left panel) andcomplementary RT-PCR analyses of TIGIT mRNA levels in resting oractivated CD25⁻ or CD25^(hi) CD4⁺ cells.

FIG. 11 provides graphs showing the fold-change in TIGIT or CD226expression on resting or activated (for one or two days) CD25, CD25⁺,CD45RA⁺, CD45RO⁺ cells, as described in Example 2(A).

FIG. 12A depicts the results of flow cytometry experiments to assess thestability of TIGIT expression on T cells, as described in Example 3.FIG. 12B depicts the results of plate-based assays to assess TIGITexpression in sorted TIGIT⁺ and TIGIT⁻ cells exposed to varyingconcentrations of anti-CD3, as described in Example 3.

FIGS. 13A-C show plots depicting the results of experiments assessingthe ability of TIGIT to modulate IL-10, IL-12p40 and IL-12p70 productionin scid mice lacking B and T cells, as described in Example 5.

FIG. 14 depicts the results of flow cytometric experiments to assessTIGIT expression on IL-17-producing versus IL-2-producing T-helpercells, as described in Example 2(A). The data in each panel isrepresentative of an experiment using PBMC from a different donor.

FIG. 15 depicts the results of mRNA analyses assessing the expressionlevels of TIGIT in disease tissue samples, as described in Example 3.The rightmost panel provides expression data from sorted cells takenfrom rheumatoid arthritis synovial tissue. PVR and CD226 expression wereundetectable in these samples.

FIG. 16 depicts the results of RT-PCR experiments assessing theexpression of TIGIT (top panel) or CD226 (lower panel) in tissues takenat various time points from mouse models of collagen-induced arthritisrelative to normal samples.

FIG. 17 depicts the results of mRNA analyses assessing the expressionlevels of TIGIT, PVR, and CD226 in tissue samples from asthmatic andcontrol rhesus monkeys, as described in Example 3.

FIG. 18A depicts the results of mRNA analyses assessing the expressionlevels of TIGIT (upper panel) in normal or cancerous cells or theexpression of CD4 in various breast tumor samples (lower panel). FIGS.18B-18D depict the results of mRNA analyses assessing the expressionlevels of TIGIT (FIG. 18B), PVR (FIG. 18C), and CD226 (FIG. 18D) invarious cancer samples, as described in Example 3. The lower panels ineach of FIGS. 18B, 18C, and 18D show levels of TIGIT, PVR, or CD226expression, respectively, in cancer samples containing variouspercentages of tumor cells. Boxes in all panels represent statisticallysignificant data.

FIGS. 19A-D depict the results of experiments assessing the effect ofTIGIT on T cell activation, as described in Example 4. FIG. 19A depictsthe results of FACS assays assessing PVR expression on CD14⁺ monocytes,iMDDC and MDDC. Anti-PVR experiments are shown without shading andisotype-matched controls are shown in grey. FIG. 19B depicts the resultsof in vitro MLR assays using TNFα-matured DC and isolated CD4⁺ T cellsassessing the effect of TIGIT-Fc on T cell proliferation. The dataindicated with the asterisk has a p<0.001. FIG. 19C depicts the resultsof experiments assessing T cell proliferation by [³H]-thymidineincorporation (cpm) (left panel) and IFN-γ production by ELISA (rightpanel) in CD4⁺ T cells activated with soluble anti-CD3 in the presenceof autologous CD11c⁺ DCs and anti-TIGIT antibody 10A7 (black bars) orisotype control (white bars). A single asterisk indicates a p<0.01; adouble asterisk indicates a p<0.001. FIG. 19D depicts the results ofexperiments assessing proliferation and IFN-γ production in naïve CD4⁺CD25⁻ T cells activated with autologous CD11c⁺ DC and soluble anti-CD3in the presence of 100 μg/mL TIGIT-Fc (grey bars) or isotype control(white bars). A single asterisk indicates a p<0.01; a double asteriskindicates a p<0.001.

FIGS. 20A and 20B depict the results of experiments assessing theability of sorted TIGIT⁺ T cells to inhibit TIGIT⁻ T cell proliferationin an MLR assay, as described in Example 4.

FIG. 21A depicts the results of proliferation assays assessing theeffect of TIGIT⁺ T_(reg) on proliferation of other T cells and APC inthe presence and absence of anti-TIGIT antibody (10A7), as described inExample 4, as well as the production of IFNγ and IL-10 in those cellpopulations. FIG. 21B depicts the results of proliferation assaysassessing the effect of TIGIT⁺ T_(regs) on naïve T cell proliferation incomparison with TIGIT⁻ T_(reg), as described in Example 4A.

FIGS. 22A-D depict the results of experiments assessing the ability ofTIGIT to modulate cytokine production in matured iMDDC and DC, asdescribed in Example 5. FIGS. 22A-1 to 22A-3 show the results of ELISAassays measuring IL-10 or IL-12p40 production in iMDDC, iMDDC stimulatedwith TNFα, iMDDC stimulated with CD40L, iMDDC stimulated with LPS, oriMDDC stimulated with Pam3CSK4. The results shown are averages fromthree experiments. Lines in each panel represent data from each of threedifferent donors. FIG. 22B shows the results of FACS analyses to measurethe expression of cell surface maturation markers HLA-DR, CD80, CD83,and CD86 in treated cells. Values are represented as mean fluorescenceintensity (MFI), and the data shown is representative of three donors.FIG. 22C shows data from experiments measuring TIGIT effects on otherproinflammatory cytokine production from TNFα-matured or LPS-maturedMDDC. The data shown are representative of three experiments. IL-6,IL12p70, and IL-18 levels were determined by LUMINEX analysis, asdescribed in Example 5. FIG. 22D shows a graph representing the relativeamounts of TGFβ secretion in iMDDC in response to TIGIT.Fc or anisotype-matched control, as described in Example 5.

FIGS. 23A-C depict the results of experiments assessing the effect ofTIGIT treatment on activation of downstream signaling by PVR, asdescribed in Example 6. FIG. 23A shows Western blot analyses of thetyrosine phosphorylation state of PVR treated with TIGIT or a control.FIG. 23B shows Western blot analyses of ERK dimerization state upontreatment of iMDDC with TIGIT-Fc, TIGIT-Fc-DANA, or control. FIG. 23Cshows Western blot analyses of active versus total β-catenin inTIGIT-treated versus control-treated iMDDC.

FIGS. 24A-B depict the results of experiments assessing the effect ofblockade of various downstream signaling molecules on TIGIT-induceddecreases in IL-12p40 production in TNFα-matured MDDC, as described inExample 6. FIG. 24A shows graphs of results from experiments testing theimpact of a MAPK kinase inhibitor on TIGIT-Fc or TIGIT-Fc-DANA-induceddecreases in IL-12p40 production. FIG. 24B shows graphs of results fromexperiments assessing the impact of an anti-TIGIT antibody (10A7), ananti-IL-10 antibody, or an anti-CD32 antibody on TIGIT-mediateddecreases in IL-12p40 production from TNFα-matured MDDC.

FIGS. 25A-B depict the results of experiments assessing the impact ofTIGIT-Fc treatment on T cell activation, as described in Example 7.Graphs of data from experiments assessing the amount of T cellproliferation (FIG. 25A) or IL-2 production (FIG. 25B) induced by/iniMDDC or TNFα/CD40L-matured MDDC cultures treated with TIGIT-Fc orcontrol antibody.

FIG. 26 depicts the results of experiments assessing the impact ofTIGIT-Fc treatment on expression of ILTs in activated human MDDC, asdescribed in Example 7.

FIGS. 27A-H depict the results of experiments assessing the effect ofTIGIT treatment on delayed type hypersensitivity responses in mice, asdescribed in Example 7. FIG. 27A shows a graph representing ear swellingdata from wild-type or IL-10 knockout mice treated with anti-ragweedantibody, TIGIT-Fc, or CTLA4. FIG. 27B shows data representing theproliferation response of spleen cells from TIGIT-Fc-, CTLA4-Fc-, orcontrol-treated mice to KLH restimulation. The data shows asresponse±standard deviation (n=3 per group; the in vitro recall assaywas performed in triplicate wells). FIG. 27C shows a graph representingear swelling data from wild-type mice treated with TIGIT-Fc,TIGIT-Fc-DANA, or anti-TIGIT antibody 10A7. FIGS. 27D and 27E depictgraphs indicating the proliferation response of spleen cells fromwild-type (FIG. 27D) or IL-10 knockout (FIG. 27E) TIGIT-Fc-treated miceto KLH restimulation. FIGS. 27F and 27G depict graphs indicating theIL-2 or IFN-γ levels in culture supernatants from splenocytes isolatedfrom wild-type (FIG. 27F) or IL-10 knockout (FIG. 27G) TIGIT-Fc-treatedmice that had been reactivated with KLH for two days. Data are shown asmean±s.d. (n=3 per group; in vitro recall was performed in triplicatewells). An asterisk indicates p<0.001. FIG. 27H depicts graphs showingthe relative mRNA levels of IL-10 (left panel), IL-12/23p40 (centerpanel), and IL-12p35 (right panel) from CD11c⁺ splenocytes of TIGIT-Fcand isotype control-treated wild-type or IL-10-deficient mice, asdetermined by qRT-PCR (n=8). IL-10 mRNA levels from WT CD11c⁻ depletedsplenocytes were also determined as a control. Data represent arbitrarymRNA levels relative to corresponding mRNA levels from unimmunized mice.An asterisk indicates p<0.05.

FIGS. 28A-28E depict the results of experiments assessing the effects ofknock-down of TIGIT expression by TIGIT-specific siRNA, as described inExample 4(B). FIG. 28A shows the results of qRT-PCR analysis of TIGITknock down efficiency versus control siRNA. CTLA4 mRNA levels weredetermined as a non-target control. FIG. 28B shows FACS analyses ofsurface TIGIT expression in siRNA_(control) and siRNA_(TIGIT)-treatedcells (summarized in Table 7). FIGS. 28C and 28D show the results ofFACS analyses of cell proliferation of CD4⁺ CD45RO⁺ human T cellsactivated with plate-bound anti-CD3 alone or in conjunction withanti-CD28 in the presence of siRNA_(control) or siRNA_(TIGIT) (FIG. 27C)or anti-TIGIT antibody 10A7 (FIG. 27D). FIG. 28E depicts the results ofanalyses of cytokine production from the cells used in the assays inFIG. 28C after two days of culture. The data shown is representative offour individual donors and experiments.

FIGS. 29A-29E depict the results of experiments assessing the expressionof CD226 on various cell types and upon various treatments. FIG. 29Adepicts the results of FACS analyses showing the surface expression ofCD226 on resting and anti-CD3 and anti-CD28 activated (day 1 and 2)sorted naïve CD4⁺CD45RA⁺ cells (top panels) or memory CD4⁺CD45RO⁺ cells(bottom panels) using anti-CD226. FIG. 29B provides graphs showing thefold-increase in mRNA levels on sorted CD4⁺CD45RO⁺, CD4⁺CD45RA⁺ and CD8⁺cells activated with anti-CD3 plus anti-CD28 for 1 or 2 days, and sortedCD56⁺ NK cells activated with IL-2 plus IL-15 for one day, as comparedto unstimulated cells. FIG. 29C shows the relative mRNA levels of avariety of cell markers on cells sorted directly ex vivo from PBMC asdetermined by qRT-PCR, as an indicator of the populations of CD4⁺, CD8⁺,CD4⁺CD45RO⁺, CD4⁺CD25^(hi)T_(regs), NK and CD11c⁺ DC cells relative tonaïve CD4⁺CD45RA⁺ cells. Data shown represents an average of data fromthree donors. FIG. 29D depicts the results of FACS analyses to determinethe co-expression of CD226 and CD25 on gated CD4⁺ cells taken from apopulation of total human PBMC stained with anti-CD4, anti-CD25, andanti-CD226. The plot shown is one representative from two donors. FIG.29E shows a graph depicting TIGIT and CD226 mRNA levels in activated andresting CD4⁺CD25⁻ and CD4⁺CD25^(hi) cells isolated from PBMC. mRNAlevels are representated as fold-change over the resting CD4⁺CD25⁻ cellsand are an average of data from two donors. FIGS. 30A-C depict theresults of experiments assessing immune cell functionality inTIGIT-deficient mice, as described in Example 8. FIG. 30A shows graphscomparing the proliferation of TIGIT-deficient (TIGIT.KO) T cells versuswild-type T cells in the absence (left panel) or presence (middle panel)of wild-type antigen-presenting cells. The right panel shows graphscomparing the proliferation of TIGIT.KO T cells to wild-type T cells inthe presence of TIGIT.KO antigen-presenting cells. FIG. 30B shows theresults of FACS assays assessing IFNγ and IL-4 levels in TIGIT.KO versuswild-type T cells. FIG. 30C are graphs showing the measured levels ofthe indicated cytokines in the supernatants of TIGIT.KO or wild-type Tcells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

TIGIT had previously been identified as a putative modulator of immunefunction (see, e.g., US patent publication no. US20040121370,incorporated herein by reference). Herein, Applicants demonstrate thatTIGIT is a member of a newly described family of immune-related proteinsthat includes poliovirus receptor (PVR, also known as NECL5 or CD155),PVR-like proteins 1-4 (PVRL1-4), CD96, and CD226. Applicants provide theconserved structural elements of this new family, whose members playroles in immune regulation and function, and provide methods to identifyfurther family members.

Applicants show that TIGIT binds tightly to PVR, and binds with lesserKd to PVRL3 (also known as nectin-3 or CD113) and PVRL2 (also known asnectin-2 or CD112). PVR is a cell surface receptor highly expressed ondendritic cells (DC), as well as FDC, fibroblasts, endothelial cells,and some tumor cells (Sakisaka, T. & Takai, Y., Curr Opin Cell Biol 16,513-21 (2004); Fuchs, A. & Colonna, M., Semin Cancer Biol 16, 359-66(2006)). Applicants show by mRNA and FACS analyses that TIGIT ispredominantly expressed on a variety of activated T cells, particularlyregulatory T cells (T_(reg)), memory T cells, NK cells, and follicular Thelper cells (T_(fh)). The studies described herein demonstrate theinteraction of TIGIT with PVR on DC, and show that this bindinginteraction modulates DC function, particularly cytokine production.TIGIT-bound human DC secreted high levels of IL-10 and fewerpro-inflammatory cytokines (such as IL-12p40 and IL-12p70). TIGITbinding to immature T cells (as assessed using TIGIT fusion constructs)inhibited T cell activation and proliferation. Notably, this inhibitionwas reversed in the presence of an ERK inhibitor, indicating that ERKactivation may be an important step in the functioning of TIGIT tomodulate DC activity. Applicants show herein that TIGIT⁺ T cellssuppress proliferation of not only other TIGIT⁻ T cells, but alsoantigen presenting cells when present in a mixed population of immunecells, and that TIGIT itself is responsible for this suppressive effect,since inclusion of a blocking anti-TIGIT antibody in the mixture greatlyreduces the observed suppression.

TIGIT is increased in expression in arthritis, psoriasis, inflammatorybowel disorder, and breast cancer tissues relative to normal controltissues, as is shown herein. Applicants also directly demonstrate theability of TIGIT to modulate immune response by showing that a TIGITfusion protein inhibited human T cell responses in vitro and murine Tcell activation in a delayed-type hypersensitivity in vivo assay. TIGITsignificantly modified mature DC, and to a lesser extent immature DC,suggesting the TIGIT-PVR interaction may be important in fine-tuning aregulatory immune response once DC become fully activatedantigen-presenting cells. The experiments presented herein suggest amechanism by which TIGIT inhibits T cell activation through aninhibitory feedback loop via the induction of IL-10 in DC. Accordingly,the invention further provides novel methods of modulating immunefunction by modulating particular subsets of cytokines or particularsubsets of immune cells. These and other aspects of the invention aredescribed in greater detail hereinbelow.

I. DEFINITIONS

The terms “TIGIT polypeptide”, “TIGIT protein” and “TIGIT” are usedinterchangeably herein and refer to specific polypeptide sequences asdescribed herein. The TIGIT polypeptides described herein may beisolated from a variety of sources, such as from human tissue or tissuefrom a nonhuman organism, or prepared by recombinant or syntheticmethods. In one embodiment, a TIGIT polypeptide has the amino acidsequence set forth in any of SEQ ID NO: 1-4. All disclosures in thisspecification which refer to the “TIGIT polypeptide” refer to each ofthe polypeptides individually as well as jointly. For example,descriptions of the preparation of, purification of, derivation of,formation of antibodies to or against, administration of, compositionscontaining, treatment of a disease with, etc., pertain to eachpolypeptide of the invention individually. The terms “TIGITpolypeptide”, “TIGIT protein”, or “TIGIT” also include variants of theTIGIT polypeptides disclosed herein or known in the art.

A “native sequence TIGIT polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding TIGIT polypeptide derivedfrom nature. Such native sequence TIGIT polypeptides can be isolatedfrom nature or can be produced by recombinant or synthetic means. Theterm “native sequence TIGIT polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific TIGITpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence TIGIT polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acid sequences. However, while theTIGIT polypeptide disclosed in the accompanying figures are shown tobegin with methionine residues designated herein as amino acid position1 in the figures, it is conceivable and possible that other methionineresidues located either upstream or downstream from the amino acidposition 1 in the figures may be employed as the starting amino acidresidue for the TIGIT polypeptides.

The TIGIT polypeptide “extracellular domain” or “ECD” refers to a formof the TIGIT polypeptide which is essentially free of the transmembraneand cytoplasmic domains. Ordinarily, a TIGIT polypeptide ECD will haveless than 1% of such transmembrane and/or cytoplasmic domains andpreferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the TIGITpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as identified herein. Optionally, therefore, anextracellular domain of a TIGIT polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary and such polypeptides, with orwithout the associated signal peptide, and nucleic acid encoding them,are contemplated by the present invention. In one embodiment, the TIGITECD encompasses amino acids 1-139 of the human TIGIT protein set forthin SEQ ID NO: 1.

The approximate locations of the “signal peptides” of the various TIGITpolypeptides disclosed herein can be identified using art-known methods.For example, the signal sequence of the human TIGIT polypeptide setforth in SEQ ID NO: 1 is predicted to span amino acids 1-15 (see, e.g.,U.S. Patent publication no. US20040121370). It is noted, however, thatthe C-terminal boundary of a signal peptide may vary, but most likely byno more than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“TIGIT polypeptide variant” means an active TIGIT polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence TIGIT polypeptide sequence asdisclosed herein, a TIGIT polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a TIGITpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length TIGIT polypeptide sequence. SuchTIGIT polypeptide variants include, for instance, TIGIT polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the full-length native amino acid sequence. Ordinarily,a TIGIT polypeptide variant will have at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence TIGIT polypeptide sequence as disclosedherein, a TIGIT polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a TIGIT polypeptide, withor without the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length TIGIT polypeptidesequence. Ordinarily, TIGIT variant polypeptides are at least about 10amino acids in length, alternatively at least about 20 amino acids inlength, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the TIGITpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific TIGIT polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is publicly available. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is alsopublicly available through Genentech, Inc., South San Francisco, Calif.The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 1 and 2 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“TIGIT”, wherein “TIGIT” represents the amino acid sequence of ahypothetical TIGIT polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“TIGIT” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

TABLE 1 Protein of interest XXXXXXXXXXXXXXX (Length = 15 amino acids)Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)% amino acid sequence identity =(the number of identically matching amino acid residues between thetwo polypeptide sequences as determined by ALIGN-2) divided by (thetotal number of amino acid residues of the protein of interest) =5 divided by 15 = 33.3%

TABLE 2 Protein of interest XXXXXXXXXX (Length = 10 amino acids)Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)% amino acid sequence identity =(the number of identically matching amino acid residues between thetwo polypeptide sequences as determined by ALIGN-2) divided by (thetotal number of amino acid residues of the protein of interest) =5 divided by 10 = 50%

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph and Tables 1 and 2 using the ALIGN-2 computerprogram. However, % amino acid sequence identity values may also beobtained as described below by using the WU-BLAST-2 computer program(Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of theWU-BLAST-2 search parameters are set to the default values. Those notset to default values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a %amino acid sequence identity value is determined by dividing (a) thenumber of matching identical amino acid residues between the amino acidsequence of the TIGIT polypeptide of interest having a sequence derivedfrom the native TIGIT polypeptide and the comparison amino acid sequenceof interest (i.e., the sequence against which the TIGIT polypeptide ofinterest is being compared which may be a TIGIT variant polypeptide) asdetermined by WU-BLAST-2 by (b) the total number of amino acid residuesof the TIGIT polypeptide of interest. For example, in the statement “apolypeptide comprising an the amino acid sequence A which has or havingat least 80% amino acid sequence identity to the amino acid sequence B”,the amino acid sequence A is the comparison amino acid sequence ofinterest and the amino acid sequence B is the amino acid sequence of theTIGIT polypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

The terms “TIGIT polynucleotide” and “TIGIT nucleotide sequence” areused interchangeably herein and refer to specific polynucleotidesequences encoding a TIGIT polypeptide. These polynucleotides maycomprise DNA or RNA or both DNA and RNA. The TIGIT polynucleotidesdescribed herein may be isolated from a variety of sources, such as fromhuman tissue or tissue from a nonhuman organism, or prepared byrecombinant or synthetic methods. All disclosures in this specificationwhich refer to a “TIGIT polynucleotide” refer to each of thepolynucleotides individually as well as jointly. For example,descriptions of the preparation of, purification of, derivation of,administration of, compositions containing, treatment of a disease with,etc., pertain to each polynucleotide of the invention individually aswell as collectively. The terms “TIGIT polynucleotide” and “TIGITnucleotide sequence” also include variants of the TIGIT polynucleotidesdisclosed herein.

A “native sequence TIGIT polynucleotide” comprises a polynucleotidehaving the same nucleic acid sequence as the corresponding TIGITpolynucleotide derived from nature. Such native sequence TIGITpolynucleotides can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence TIGITpolynucleotide” specifically encompasses polynucleotides encodingnaturally-occurring truncated or secreted forms of the specific TIGITpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence TIGIT polynucleotidesdisclosed herein are mature or full-length native sequencepolynucleotides comprising the full-length nucleic acid sequences.

A “TIGIT variant polynucleotide” or “TIGIT variant nucleic acidsequence” means a nucleic acid molecule which encodes an active TIGITpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence TIGIT polypeptide sequence as disclosedherein, a full-length native sequence TIGIT polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of aTIGIT polypeptide, with or without the signal peptide, as disclosedherein or any other fragment of a full-length TIGIT polypeptidesequence. Ordinarily, a TIGIT variant polynucleotide will have at leastabout 80% nucleic acid sequence identity, alternatively at least about81% nucleic acid sequence identity, alternatively at least about 82%nucleic acid sequence identity, alternatively at least about 83% nucleicacid sequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence TIGIT polypeptide sequence, a full-length nativesequence TIGIT polypeptide sequence lacking the signal peptide, anextracellular domain of a TIGIT polypeptide, with or without the signalsequence, or any other fragment of a full-length TIGIT polypeptidesequence. Variants do not encompass the native nucleotide sequence.

Ordinarily, TIGIT variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toTIGIT-encoding nucleic acid sequences identified herein is defined asthe percentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the TIGIT nucleic acid sequence of interest,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. The ALIGN-2 sequence comparisoncomputer program was authored by Genentech, Inc. and the source code hasbeen filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the publicly available source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 3 and 4, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “TIGIT-DNA”,wherein “TIGIT-DNA” represents a hypothetical TIGIT-encoding nucleicacid sequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “TIGIT-DNA”nucleic acid molecule of interest is being compared, and “N”, “L” and“V” each represent different hypothetical nucleotides.

TABLE 3 DNA of interest NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)% nucleic acid sequence identity =(the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the DNA of interest) =6 divided by 14 = 42.9%

TABLE 4 DNA of interest NNNNNNNNNNNN (Length = 12 nucleotides)Comparison DNA NNNNLLLVV (Length = 9 nucleotides)% nucleic acid sequence identity =(the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the DNA of interest) =4 divided by 12 = 33.3%

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph and Tables 3 and 4 using the ALIGN-2 computerprogram. However, % nucleic acid sequence identity values may also beobtained as described below by using the WU-BLAST-2 computer program(Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of theWU-BLAST-2 search parameters are set to the default values. Those notset to default values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a %nucleic acid sequence identity value is determined by dividing (a) thenumber of matching identical nucleotides between the nucleic acidsequence of the TIGIT polypeptide-encoding nucleic acid molecule ofinterest having a sequence derived from the native sequence TIGITpolypeptide-encoding nucleic acid and the comparison nucleic acidmolecule of interest (i.e., the sequence against which the TIGITpolypeptide-encoding nucleic acid molecule of interest is being comparedwhich may be a variant TIGIT polynucleotide) as determined by WU-BLAST-2by (b) the total number of nucleotides of the TIGIT polypeptide-encodingnucleic acid molecule of interest. For example, in the statement “anisolated nucleic acid molecule comprising a nucleic acid sequence Awhich has or having at least 80% nucleic acid sequence identity to thenucleic acid sequence B”, the nucleic acid sequence A is the comparisonnucleic acid molecule of interest and the nucleic acid sequence B is thenucleic acid sequence of the TIGIT polypeptide-encoding nucleic acidmolecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, TIGIT variant polynucleotides are nucleic acidmolecules that encode an active TIGIT polypeptide and which are capableof hybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length TIGITpolypeptide as disclosed herein. TIGIT variant polypeptides may be thosethat are encoded by a TIGIT variant polynucleotide.

“Isolated,” when used to describe the various polypeptides disclosedherein, means a polypeptide that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” TIGIT polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-TIGIT monoclonal antibodies orantibodies that specifically bind to any of the other polypeptidesdescribed herein (including agonist, antagonist, and neutralizingantibodies), anti-TIGIT or antibody compositions with polyepitopicspecificity, single chain anti-TIGIT or other antibodies, and fragmentsof anti-TIGIT or other antibodies (see below). The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide of interest (as one nonlimitingexample, a TIGIT polypeptide) fused to a “tag polypeptide”. The tagpolypeptide has enough residues to provide an epitope against which anantibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of apolypeptide (as a nonlimiting example, a TIGIT polypeptide) which retaina biological and/or an immunological activity of native ornaturally-occurring form of that polypeptide (in the previous example, aTIGIT activity), wherein “biological” activity refers to a biologicalfunction (either inhibitory or stimulatory) caused by a native ornaturally-occurring polypeptide other than the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring polypeptide and an “immunological”activity refers to the ability to induce the production of an antibodyagainst an antigenic epitope possessed by a native ornaturally-occurring polypeptide (in the previous example, a TIGITantigenic epitope).

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a TIGIT polypeptide,or to a molecule in a signaling pathway that modulates the expression ofTIGIT. The generation and therapeutic use of aptamers are wellestablished in the art. See, e.g., U.S. Pat. No. 5,475,096, and thetherapeutic efficacy of Macugen® (Eyetech, New York) for treatingage-related macular degeneration.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativepolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of apolypeptide may comprise contacting a polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the polypeptide.

The terms “TIGIT antagonist” and “antagonist of TIGIT activity or TIGITexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of TIGIT, either by decreasingtranscription or translation of TIGIT-encoding nucleic acid, or byinhibiting or blocking TIGIT polypeptide activity, or both. Examples ofTIGIT antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragmentsof anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-bindingpeptides, and other polypeptides that specifically bind TIGIT(including, but not limited to, TIGIT-binding fragments of one or moreTIGIT ligands, optionally fused to one or more additional domains), suchthat the interaction between the TIGIT antagonist and TIGIT results in areduction or cessation of TIGIT activity or expression. It will beunderstood by one of ordinary skill in the art that in some instances, aTIGIT antagonist may antagonize one TIGIT activity without affectinganother TIGIT activity. For example, a desirable TIGIT antagonist foruse in certain of the methods herein is a TIGIT antagonist thatantagonizes TIGIT activity in response to one of PVR interaction, PVRL3interaction, or PVRL2 interaction, e.g., without affecting or minimallyaffecting any of the other TIGIT interactions.

The terms “PVR antagonist” and “antagonist of PVR activity or PVRexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of PVR, either by decreasingtranscription or translation of PVR-encoding nucleic acid, or byinhibiting or blocking PVR polypeptide activity, or both. Examples ofPVR antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,PVR-specific aptamers, anti-PVR antibodies, PVR-binding fragments ofanti-PVR antibodies, PVR-binding small molecules, PVR-binding peptides,and other polypeptides that specifically bind PVR (including, but notlimited to, PVR-binding fragments of one or more PVR ligands, optionallyfused to one or more additional domains), such that the interactionbetween the PVR antagonist and PVR results in a reduction or cessationof PVR activity or expression. It will be understood by one of ordinaryskill in the art that in some instances, a PVR antagonist may antagonizeone PVR activity without affecting another PVR activity. For example, adesirable PVR antagonist for use in certain of the methods herein is aPVR antagonist that antagonizes PVR activity in response to TIGITinteraction without impacting the PVR-CD96 and/or PVR-CD226interactions.

The terms “TIGIT agonist” and “agonist of TIGIT activity or TIGITexpression” are used interchangeably and refer to a compound thatenhances or stimulates the normal functioning of TIGIT, by increasingtranscription or translation of TIGIT-encoding nucleic acid, and/or byinhibiting or blocking activity of a molecule that inhibits TIGITexpression or TIGIT activity, and/or by enhancing normal TIGIT activity(including, but not limited to, enhancing the stability of TIGIT orenhancing binding of TIGIT to one or more target ligands). For example,the TIGIT agonist can be selected from an antibody, an antigen-bindingfragment, an aptamer, an interfering RNA, a small molecule, a peptide,an antisense molecule, and another binding polypeptide. In anotherexample, the TIGIT agonist can be a polynucleotide selected from anaptamer, interfering RNA, or antisense molecule that interferes with thetranscription and/or translation of a TIGIT-inhibitory molecule. It willbe understood by one of ordinary skill in the art that in someinstances, a TIGIT agonist may agonize one TIGIT activity withoutaffecting another TIGIT activity. For example, a desirable TIGIT agonistfor use in certain of the methods herein is a TIGIT agonist thatagonizes TIGIT activity in response to one of PVR interaction, PVRL3interaction, or PVRL2 interaction, e.g., without affecting or minimallyaffecting any of the other TIGIT interactions.

The terms “PVR agonist” and “agonist of PVR activity or PVR expression”are used interchangeably and refer to a compound that enhances orstimulates the normal functioning of PVR, by increasing transcription ortranslation of PVR-encoding nucleic acid, and/or by inhibiting orblocking activity of a molecule that inhibits PVR expression or PVRactivity, and/or by enhancing normal PVR activity (including, but notlimited to, enhancing the stability of PVR or enhancing binding of PVRto one or more target ligands). For example, the PVR agonist can beselected from an antibody, an antigen-binding fragment, an aptamer, aninterfering RNA, a small molecule, a peptide, an antisense molecule, andanother binding polypeptide. In another example, the PVR agonist can bea polynucleotide selected from an aptamer, interfering RNA, or antisensemolecule that interferes with the transcription and/or translation of aPVR-inhibitory molecule. It will be understood by one of ordinary skillin the art that in some instances, a PVR agonist may agonize one PVRactivity without affecting another PVR activity. For example, adesirable PVR agonist for use in certain of the methods herein is a PVRagonist that agonizes PVR activity in response to TIGIT interaction, orwhich mimics TIGIT in interacting with PVR, e.g., without affecting orminimally affecting PVR-CD96 or PVR-CD226 binding interactions.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing a dye or stain such as, but not limited to, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced using certainprocedures known in the art. For example, Marks et al. Bio/Technology10:779-783 (1992) describes affinity maturation by VH and VL domainshuffling. Random mutagenesis of HVR and/or framework residues isdescribed by, for example, Barbas et al. Proc Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton etal. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896(1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics at least one of the functional activities of a polypeptideof interest.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include, but are not limited to, those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a polypeptide described herein or antibody thereto) to amammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “immune-related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means an immune-related disease inwhich T cells directly or indirectly mediate or otherwise contribute toa morbidity in a mammal. The T cell mediated disease may be associatedwith cell mediated effects, lymphokine mediated effects, etc., and eveneffects associated with B cells if the B cells are stimulated, forexample, by the lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, which can be treated according to theinvention include systemic lupus erythematosis, rheumatoid arthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disorder (IBD)(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. Infectiousdiseases including viral diseases such as AIDS (HIV infection),hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungalinfections, protozoal infections and parasitic infections also may haveimmune and/or inflammatory components and/or etiology.

Several diseases of the skin are correlated with an aberrant immuneresponse and to autoimmunity. Diseases such as psoriasis are hallmarkedby skin blistering, skin flaking, edema and the presence ofautoantibodies that bind to skin proteins. In this application,experiments determine that TIGIT expression is upregulated in psoriaticskin vs. normal skin. Modulation of TIGIT expression and/or activity maybe useful in treating the symptoms or underlying causes of psoriasis.

The term inflammatory bowel disorder (“IBD”) describes a group ofchronic inflammatory disorders of unknown causes in which the intestine(bowel) becomes inflamed, often causing recurring cramps or diarrhea.The prevalence of IBD in the US is estimated to be about 200 per 100,000population. Patients with IBD can be divided into two major groups,those with ulcerative colitis (“UC”) and those with Crohn's disease(“CD”).

In patients with UC, there is an inflammatory reaction primarilyinvolving the colonic mucosa. The inflammation is typically uniform andcontinuous with no intervening areas of normal mucosa. Surface mucosalcells as well as crypt epithelium and submucosa are involved in aninflammatory reaction with neutrophil infiltration. Ultimately, thissituation typically progresses to epithelial damage with loss ofepithelial cells resulting in multiple ulcerations, fibrosis, dysplasiaand longitudinal retraction of the colon. CD differs from UC in that theinflammation extends through all layers of the intestinal wall andinvolves mesentery as well as lymph nodes. CD may affect any part of thealimentary canal from mouth to anus. The disease is often discontinuous,i.e., severely diseased segments of bowel are separated from apparentlydisease-free areas. In CD, the bowel wall also thickens which can leadto obstructions. In addition, fistulas and fissures are not uncommon.

Clinically, IBD is characterized by diverse manifestations oftenresulting in a chronic, unpredictable course. Bloody diarrhea andabdominal pain are often accompanied by fever and weight loss. Anemia isnot uncommon, as is severe fatigue. Joint manifestations ranging fromarthralgia to acute arthritis as well as abnormalities in liver functionare commonly associated with IBD. Patients with IBD also have anincreased risk of colon carcinomas compared to the general population.During acute “attacks” of IBD, work and other normal activity areusually impossible, and often a patient is hospitalized.

Although the cause of IBD remains unknown, several factors such asgenetic, infectious and immunologic susceptibility have been implicated.IBD is much more common in Caucasians, especially those of Jewishdescent. The chronic inflammatory nature of the condition has promptedan intense search for a possible infectious cause. Although agents havebeen found which stimulate acute inflammation, none has been found tocause the chronic inflammation associated with IBD. The hypothesis thatIBD is an autoimmune disease is supported by the previously mentionedextraintestinal manifestation of IBD as joint arthritis, and the knownpositive response to IBD by treatment with therapeutic agents such asadrenal glucocorticoids, cyclosporine and azathioprine, which are knownto suppress immune response. In addition, the GI tract, more than anyother organ of the body, is continuously exposed to potential antigenicsubstances such as proteins from food, bacterial byproducts (LPS), etc.

Further, the risk of colon cancer is highly elevated in patients withsevere ulcerative colitis, particularly if the disease has existed forseveral years. About 20-25% of patients with IBD eventually requiresurgery for removal of the colon because of massive bleeding, chronicdebilitating illness, performation of the colon, or risk of cancer.Surgery is also sometimes performed when other forms of medicaltreatment fail or when the side effects of steroids or other medicationsthreaten the patient's health. As surgery is invasive and drasticallylife altering, it is not a highly desirable treatment regimen, and istypically the treatment of last resort. In order to better understandthis disease and possibly treat it, experiments determined that TIGITwas upregulated both in CD and UC when compared to normal tissue.Modulation of the expression and/or activity of TIGIT may prove usefulin the treatment of one or more forms of IBD.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self IgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid if infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, intestitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrhematoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rhematoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

The term “effective amount” is a concentration or amount of apolypeptide and/or agonist/antagonist which results in achieving aparticular stated purpose. An “effective amount” of a polypeptide oragonist or antagonist thereof may be determined empirically.Furthermore, a “therapeutically effective amount” is a concentration oramount of a polypeptide and/or agonist/antagonist which is effective forachieving a stated therapeutic effect. This amount may also bedetermined empirically.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,caminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found, for example, in TheMolecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,entitled “Cell cycle regulation, oncogens, and antineoplastic drugs” byMurakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators. Certainexamples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are, e.g., growthhormone such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-13;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG1,IgG2, IgG3, or IgG4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgDor IgM.

As used herein, the term “inflammatory cells” designates cells thatenhance the inflammatory response such as mononuclear cells,eosinophils, macrophages, and polymorphonuclear neutrophils (PMN).

II. COMPOSITIONS AND METHODS OF THE INVENTION

TIGIT had previously been identified as a putative modulator of immunefunction (see, e.g., US patent publication no. US20040121370,incorporated herein by reference). Herein, Applicants demonstrate thatTIGIT is a member of a newly described family of immune-related proteinstermed the “TIGIT-like protein” (TLP) family that includes poliovirusreceptor (PVR, also known as NECL5 or CD155), PVR-like proteins 1-4(PVRL1-4), CD96, and CD226. Applicants provide the conserved structuralelements of this new TLP family, whose members play roles in immuneregulation and function, and provide methods to identify further familymembers. PVRL1-4 and PVR share a common domain architecture(IgV-IgC-IgV), whereas CD226 and CD96 lack the membrane proximal IgVdomain. The intracellular segments of these eight proteins show only alimited similarity with each other outside of the afadin binding motifshared between PVRL1-3; PVRL4 lacks this sequence but still is known tobind afadin. Based on the crystal structure of the related IgV domain ofNECL-1 (Dong et al., J. Biol. Chem. 281: 10610-17 (2006)) the first andthird motifs are predicted to lie in hairpin loops between the B and Cand the F and G beta-strands, respectively. These two loops are adjacentto each other at one end of the IgV fold. The second motif comprises theC′ and C″ beta-strands that are involved in forming part of thehomodimeric interface for NECL-1. Thus, these sequence motifs may play arole in specific homo- and heterotypic interactions observed between PVRfamily members.

The TLP family members comprise a number of absolutely conserved aminoacids, including alanine⁶⁷, glycine⁷⁴, proline¹¹⁴, and glycine¹¹⁶.Additionally, TLP family members comprise several amino acids which aresubstantially conserved (e.g., found in the majority of family members,but not in every family member), including an amino acid selected fromvaline, isoleucine, and leucine at position 54, an amino acid selectedfrom serine and threonine at position 55, a glutamine at position 56, athreonine at position 112, and an amino acid selected from phenylalanineand tyrosine at position 113. Members of the TLP family also comprisethree structural submotifs:valine/isoleucine⁵⁴-serine/threonine⁵⁵-glutamine⁵⁶;alanine⁶⁷-X⁶⁸⁻⁷³-glycine⁷⁴ (where X is any amino acid); andthreonine¹¹²-phenylalanine/tyrosine¹¹³-proline¹¹⁴-x¹¹⁵-glycine¹¹⁶ (whereX is any amino acid). It will be understood by one of ordinary skill inthe art that the numbering used above is with respect to the human TIGITprotein sequence, and while the relative position of these conservedresidues and motifs in different members of the TLP protein family areidentical to the position of those amino acids in the human TIGITsequence, the absolute numbering of those residues in other TLP familymembers may differ.

Given the involvement of the identified TLP family members in immuneregulation and function, other members of this protein family are alsolikely to be involved in immune regulation and function. Accordingly,the invention provides methods of determining whether a given protein isa member of the TLP family by aligning the sequence of the protein tothe sequences of one or more of the above-identified family members andassessing the presence or absence in the given protein sequence of theabove-identified absolutely conserved residues, the above-identifiedsubstantially conserved residues, and/or the above-identified structuralsubmotifs. The invention also provides methods of identifying othermembers of the TLP protein family by searching one or more sequencedatabases for proteins whose amino acid sequences comprise theabove-identified absolutely conserved residues, the above-identifiedsubstantially conserved residues, and/or the above-identified structuralsubmotifs.

The identification of the TLP family by Applicants herein also presentsthe possibility that the common structural features of the TLP familymembers may permit two or more members of the TLP family to be similarlymodulated. For example, if the conserved and substantially conservedamino acid residues and submotifs in each TLP family member give rise tosimilar three-dimensional structures in those family members in one ormore domains of each protein, then those similar three-dimensionalstructures may be targeted in order to simultaneously modulate more thanone TLP family member, or even all TLP family members at the same time.The invention thus also provides agents (“TLP-interacting agents”) thatspecifically interact with such conserved or substantially conservedregions of TLP family members. Such agents may be used to identify oneor more further members of the TLP family by assessing whether acandidate protein interacts with a TLP-interacting agent. Interaction ofthe candidate protein with the TLP-interacting agent may indicate thatthe protein may also be a TLP family member. TLP-interacting agents maymodulate TLP activity. For example, a TLP-interacting agent may be anantagonist of TLP activity, including, but not limited to, a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, and an inhibitory peptide. In another example, aTLP-interacting agent may be an agonist of TLP activity, including, butnot limited to, an agonizing antibody or antigen-binding fragmentthereof, an agonizing peptide, and a small molecule that stabilizes aTLP protein structure to facilitate TLP protein activity.TLP-interacting agents may be identified in a variety of art-known ways,for example by using the screening methods described herein.

Applicants show by mRNA and FACS analyses that TIGIT is predominantlyexpressed on a variety of activated T cells, particularly regulatory Tcells (T_(reg)), memory T cells, NK cells, and follicular B cell helperT cells (T_(fh)) isolated from tonsillar tissue. The invention thusprovides methods of identifying whether or not a selected cell is aT_(reg), memory T cell, NK cell, or T_(Fh) cell based on whether or notthe cell expresses TIGIT. The invention also provides methods of usingTIGIT to purify T_(reg), memory T cells, NK cells, and T_(Fh) cells awayfrom other types of immune cells that do not express TIGIT using any ofthe purification methods known in the art and/or described herein (asone nonlimiting example, by flow cytometry). Applicants also demonstratethat the highest expression of TIGIT in these cell populations occurs inactivated T_(regs). Thus, the invention also provides methods ofidentifying whether a given cell is an activated T_(reg) based on itsexpression level of TIGIT relative to TIGIT expression levels in one ormore control samples (where the control samples may be predeterminedvalues from exemplary T cell subset populations, or the control samplesmay be other samples from known T cell subpopulations such as activatedT_(reg), unactivated T_(reg), naïve T cells, memory T cells, NK cells,T_(Fh) cells, or other T cell populations). Also provided are methods ofdetermining whether a given T_(reg) cell is activated, by determiningits expression level of TIGIT relative to TIGIT expression levels in oneor more control activated or unactivated T_(reg) samples or relative topredetermined TIGIT expression values in known activated or unactivatedT_(reg) cell populations. Further provided are methods of separatelyisolating activated T_(reg) from other T cells using any of thepurification methods known in the art and/or described herein where thequantity of TIGIT expressed in the cell can be used to separate the cellfrom other cells (as one nonlimiting example, by flow cytometry).

Applicants demonstrate herein that TIGIT binds tightly to PVR, and bindswith lesser Kd to PVRL3 (also known as nectin-3 or CD113) and PVRL2(also known as nectin-2 or CD112). As exemplified by Applicants, TIGITbinding to PVR blocks the interaction of PVR with two other ligands,CD226 and CD96, and CD226 is a less effective inhibitor of the TIGIT-PVRinteraction than TIGIT is of the PVR-CD226 interaction. Applicantsproduced anti-TIGIT antibodies (for example, the anti-TIGIT antibody10A7 described herein) which inhibited the binding of TIGIT or a TIGITfusion protein to cell surface-expressed PVR. Applicants furtherproduced other antibodies, such as the antibody 1F4 described herein,with different epitope specificities on TIGIT than 10A7. Notably, CD226is not significantly expressed in T_(regs) or T_(Fh), two cell typesthat highly express TIGIT.

Supported by these findings, the invention provides agonists andantagonists of the TIGIT-PVR interaction, the TIGIT-PVRL2 interaction,and the TIGIT-PVRL3 interaction, and methods of modulating TIGIT-PVRbinding, TIGIT-PVRL2 binding and TIGIT-PVRL3 binding in vitro or in vivousing such agonists and antagonists. Also provided are methods ofmodulating the CD226-PVR interaction and/or the CD96-PVR interaction byadministering TIGIT (a competitor for PVR binding) or an anti-TIGITantibody or antigen-binding fragment thereof in vitro or in vivo. Theinvention further includes anti-TIGIT antibodies and fragments thereof,both agonizing and antagonizing, and in particular anti-TIGIT antibodies10A7 and 1F4 and alternate types of antibodies comprising the CDRs ofanti-TIGIT antibody 10A7 and/or 1F4.

The studies described herein demonstrate the interaction of TIGIT withPVR on DC, and show that this binding interaction modulates DC function,particularly cytokine production. PVR is a cell surface receptor knownto be highly expressed on dendritic cells (DC), as well as FDC,fibroblasts, endothelial cells, and some tumor cells (Sakisaka, T. &Takai, Y., Curr Opin Cell Biol 16, 513-21 (2004); Fuchs, A. & Colonna,M., Semin Cancer Biol 16, 359-66 (2006)). TIGIT-bound human DC secretedhigh levels of IL-10 and fewer pro-inflammatory and other cytokines(such as IL-12p40, IL-12p70, IL-6, IL-18, and IFNγ). TIGIT had no effecton production of certain cytokines such as IL-23. This cytokine skewingupon TIGIT binding was only observed in cells that had been stimulatedby TNFα or CD40/LPS, and not in TLR2- or Pam3CSK4-stimulated cells,suggesting that TIGIT is one means by which the immune system mayfine-tune DC function. TIGIT binding to immature T cells (as assessedusing TIGIT fusion constructs) inhibited T cell activation andproliferation. However, TIGIT treatment did not affect the ability ofimmature monocyte-derived DC (iMDDC) to mature, nor did it directlyinduce maturation of those cells. Notably, this inhibition was reversedin the presence of an ERK inhibitor, indicating that ERK activation maybe an important step in the functioning of TIGIT to modulate DCactivity. In fact, Applicants demonstrate that binding of TIGIT to PVRresults in phosphorylation of PVR and increased phosphorylation of pERKdimer but not pERK monomer. This was not a generalized effect, since,for example, the p38 intracellular signaling pathway was not modulatedby TIGIT-Fc treatment of cells. Applicants show herein that TIGIT⁺ Tcells suppress proliferation of not only other TIGIT⁻ T cells, but alsoantigen presenting cells when present in a mixed population of immunecells. Applicants further demonstrate that the TIGIT-PVR interactionmediates the above observed effects, since inclusion of an anti-TIGITantibody or an anti-PVR antibody in the experiments greatly reduced theobserved inhibition of proliferation, modulation of DC cytokineproduction, and suppression of proliferation of other immune cells.Overall, the data provided by Applicants herein suggests that TIGITprovides an immune system feedback mechanism by negatively regulatingimmune response.

Accordingly, the invention provides methods of modulating immune cell(e.g., DC) function by modulating TIGIT or PVR expression and/oractivity. For example, methods are provided for decreasing or inhibitingproliferation of immune cells (for example, DC or antigen-presentingcells) by treating immune cells in vitro or in vivo with TIGIT, anagonist of TIGIT expression and/or activity, or an agonist of PVRexpression and/or activity. Methods are also provided for increasingproliferation of immune cells (for example, DC or antigen-presentingcells) by treating immune cells in vitro or in vivo with an antagonistof TIGIT expression and/or activity or an antagonist of PVR expressionand/or activity. The invention also provides methods forincreasing/stimulating an immune response by administering an antagonistof TIGIT expression and/or activity or an antagonist of PVR expressionand/or activity. Similarly provided are methods fordecreasing/inhibiting an immune response by administering TIGIT, anagonist of TIGIT expression and/or activity or an agonist of PVRexpression and/or activity.

Also provided by the invention are methods of modulating the type and/oramount of cytokine production from an immune cell (e.g., DC) bymodulating TIGIT or PVR expression and/or activity. Specifically, theinvention provides methods of increasing IL-10 production by immunecells, for example DC, by treating cells in vitro or in vivo with TIGIT,an agonist of TIGIT expression and/or activity, or an agonist of PVRexpression and/or activity. Also provided are methods of decreasingproinflammatory cytokine production and/or release by immune cells, forexample DC, by treating cells in vitro or in vivo with TIGIT, an agonistof TIGIT expression and/or activity, or an agonist of PVR expressionand/or activity. Similarly, methods of decreasing IL-10 production byimmune cells, for example DC, by treating cells in vitro or in vivo withan antagonist of TIGIT expression and/or activity or an antagonist ofPVR expression and/or activity are also provided. The invention furtherprovides methods of increasing proinflammatory cytokine productionand/or release by immune cells, for example, DC, by treating cells invitro or in vivo with an antagonist of TIGIT expression and/or activityor an antagonist of PVR expression and/or activity. Also provided aremethods of stimulating ERK phosphorylation and/or intracellularsignaling through the ERK pathway in one or more cells by treating thecells with TIGIT, an agonist of TIGIT expression and/or activity, or anagonist of PVR expression and/or activity. Similarly, the inventionprovides methods of inhibiting or decreasing ERK phosphorylation and/orintracellular signaling through the ERK pathway in one or more cells bytreating the cells with an antagonist of TIGIT expression and/oractivity or an antagonist of PVR expression and/or activity.

TIGIT is increased in expression in arthritis, psoriasis, inflammatorybowel disorder, and breast cancer tissues relative to normal controltissues, as is shown herein. With regard to the breast cancer tissues,Applicants show that TIGIT expression does not correlate with tumorcells per se, but rather with CD4⁺ immune cell infiltrates in tumors.Applicants also directly demonstrate the ability of TIGIT to modulateimmune response by showing that a TIGIT fusion protein inhibited human Tcell responses in vitro and murine T cell activation in a delayed-typehypersensitivity in vivo assay. Accordingly, the invention providesmethods of diagnosing diseases/disorders involving aberrant immune cellresponse in a subject by assessing the expression and/or activity ofTIGIT in a sample from the subject and comparing the expression and/oractivity to a reference amount of TIGIT expression and/or activity orthe amount of TIGIT expression and/or activity in a sample from a normalsubject. The invention also provides methods of assessing the severityof a disease or disorder involving aberrant immune cell response (i.e.,an immune-related disease) in a subject by assessing the expressionand/or activity of TIGIT in a sample from the subject and comparing theexpression and/or activity to a reference amount of TIGIT expressionand/or activity or the amount of TIGIT expression and/or activity in asample from a normal subject. Also provided are methods of preventing adisease or disorder involving aberrant immune cell response (i.e., animmune-related disease) by modulating TIGIT expression and/or activity.Further provided are methods of treating or lessening the severity of adisease or disorder involving aberrant immune cell response (i.e., animmune-related disease) by modulating TIGIT expression and/or activity.Modulation of TIGIT expression and/or activity may take the form ofinhibiting TIGIT activity and/or expression (i.e., with a TIGITantagonist or a PVR antagonist) when the negative regulatory activitiesof TIGIT are contributing to the disease state. For example,antagonizing TIGIT expression and/or activity may be desirable when anincrease in proliferation of DC and/or increased production ofproinflammatory cytokines by DC is desirable. Modulation of TIGITexpression and/or activity may take the form of activating or increasingTIGIT expression and/or activity (i.e., by administering TIGIT, a TIGITagonist or a PVR agonist) when the negative regulatory activities ofTIGIT are desirable to control a disease state. For example, agonizingTIGIT expression and/or activity may be desirable when a decrease inproliferation of DC and/or decreased release of proinflammatorycytokines by DC is desirable. These and other aspects of the inventionare described in greater detail hereinbelow.

A. Full-Length TIGIT Polypeptides

The present invention provides isolated nucleotide sequences encodingpolypeptides referred to in the present application as TIGITpolypeptides. In particular, cDNAs encoding various TIGIT polypeptideshave been identified and isolated, as disclosed in further detail in thespecification and Examples below. It will be understood by one ofordinary skill in the art that the invention also provides otherpolypeptides useful in the methods of the invention (i.e., PVR) and thatany of the description herein drawn specifically to the method ofcreation, production, labeling, posttranslational modification, use orother aspects of TIGIT polypeptides will also be applicable to othernon-TIGIT polypeptides.

B. TIGIT Polypeptide Variants

In addition to the full-length native sequence TIGIT polypeptidesdescribed herein, it is contemplated that TIGIT variants can beprepared. TIGIT variants can be prepared by introducing appropriatenucleotide changes into the TIGIT polynucleotide, and/or by synthesis ofthe desired TIGIT polypeptide. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of theTIGIT, such as changing the number or position of glycosylation sites oraltering the membrane anchoring characteristics of the polypeptide.

Variations in the native full-length sequence TIGIT or in variousdomains of the TIGIT described herein, can be made, for example, usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion and/or insertionof one or more codons encoding the TIGIT that results in a change in theamino acid sequence of the TIGIT as compared with the native sequenceTIGIT. Optionally, the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe TIGIT. Guidance in determining which amino acid residues may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the TIGIT with thatof homologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

TIGIT polypeptide fragments are also provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the TIGIT polypeptide.

TIGIT fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating TIGIT fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, TIGIT polypeptide fragments share atleast one biological and/or immunological activity with the native TIGITpolypeptide disclosed herein.

In certain embodiments, conservative substitutions of interest are shownin Table 5 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 5, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 5 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val lys; gln; asn lys Arg (R) gln; his; lys; arggln Asn (N) glu Asp (D) glu ser Cys (C) ser asn Gln (Q) asn asp Glu (E)asp ala Gly (G) pro; ala arg His (H) Ile (I) asn; gln; lys; arg leu leu;val; met; ala; phe; Leu (L) norleucine ile norleucine; ile; val; arg Lys(K) met; ala; phe leu Met (M) arg; gln; asn leu Phe (F) leu; phe; ileala Pro (P) leu; val; ile; ala; tyr thr Ser (S) ala ser Thr (T) thr tyrTT (W) ser phe Tyr (Y) tyr; phe Val (V) leu trp; phe; thr; ser ile; leu;met; phe; ala; norleucine

Substantial modifications in function or immunological identity of thepolypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gln, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of TIGIT

Covalent modifications of TIGIT are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a polypeptide with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues of the TIGIT polypeptide. Derivatization withbifunctional agents is useful, for instance, for crosslinking TIGITpolypeptide to a water-insoluble support matrix or surface for use inthe method for purifying anti-TIGIT antibodies, and vice-versa. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the TIGIT polypeptides includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in a native sequence TIGIT(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceTIGIT. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present. Additionof glycosylation sites to a polypeptide may be accomplished by alteringthe amino acid sequence. The alteration may be made, for example, by theaddition of, or substitution by, one or more serine or threonineresidues to the native sequence polypeptide (for O-linked glycosylationsites). The polypeptide's amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are described in the art, e.g., in WO 87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of a polypeptide disclosed hereincomprises linking the polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The polypeptides of the present invention may also be modified in a wayto form a chimeric molecule comprising a polypeptide fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thepolypeptide of interest with a tag polypeptide which provides an epitopeto which an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the polypeptideof interest. The presence of such epitope-tagged forms of thepolypeptide of interest can be detected using an antibody against thetag polypeptide. Also, provision of the epitope tag enables thepolypeptide of interest to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include, but are not limited to, theFlag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3epitope peptide [Martin et al., Science, 255:192-194 (1992)]; analpha-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the polypeptide with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a polypeptide in place of at least one variable region within anIg molecule. In one embodiment, the immunoglobulin fusion includes thehinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1molecule. For the production of immunoglobulin fusions see also U.S.Pat. No. 5,428,130 issued Jun. 27, 1995.

D. Polypeptide Preparation

The description below relates primarily to production of polypeptides byculturing cells transformed or transfected with a vector containingnucleic acid encoding the polypeptide of interest. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare polypeptides. For instance, the polypeptidesequence, or portions thereof, may be produced by direct peptidesynthesis using solid-phase techniques [see, e.g., Stewart et al.,Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif.(1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the polypeptide may bechemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length polypeptide.

1. Isolation of DNA Encoding the Polypeptide

DNA encoding a polypeptide of interest may be obtained from a cDNAlibrary prepared from tissue believed to possess the polypeptide mRNAand to express it at a detectable level. Accordingly, human DNA encodingthe polypeptide can be conveniently obtained from a cDNA libraryprepared from human tissue. The polypeptide-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to thepolypeptide or oligonucleotides of at least about 20-80 bases) designedto identify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding the polypeptide is to use PCR methodology [Sambrook et al.,supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated polypeptide arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/−DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding polypeptide may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The polypeptide may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the polypeptide-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion thesignal sequence may be, e.g., the yeast invertase leader, alpha factorleader (including Saccharomyces and Kluyveromyces α-factor leaders, thelatter described in U.S. Pat. No. 5,010,182), or acid phosphataseleader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr.1990), or the signal described in WO 90/13646 published 15 Nov. 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

Examples of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thepolypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the polypeptide-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingpolypeptides.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Polypeptide transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the polypeptide by higher eukaryotes maybe increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide coding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of a polypeptide of interest in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620-625(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencepolypeptide or against a synthetic peptide based on the DNA sequencesprovided herein or against exogenous sequence fused to DNA encoding thepolypeptide and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of a polypeptide of interest may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released fromthe membrane using a suitable detergent solution (e.g. Triton-X 100) orby enzymatic cleavage. Cells employed in expression of the polypeptidecan be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify the polypeptide from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the polypeptide. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularpolypeptide produced.

E. Tissue Distribution

The location of tissues expressing the polypeptide can be identified bydetermining mRNA expression in various human tissues. The location ofsuch genes provides information about which tissues are most likely tobe affected by the stimulating and inhibiting activities of thepolypeptides. The location of a gene in a specific tissue also providessample tissue for the activity blocking/activating assays discussedbelow.

As noted before, gene expression in various tissues may be measured byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205[1980]), dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof a polypeptide or against a synthetic peptide based on the DNAsequences encoding the polypeptide or against an exogenous sequencefused to a DNA encoding a polypeptide and encoding a specific antibodyepitope. General techniques for generating antibodies, and specialprotocols for Northern blotting and in situ hybridization are providedbelow.

F. Antibody Binding Studies

The activity of a polypeptide of the invention can be further verifiedby antibody binding studies, in which the ability of anti-polypeptideantibodies to inhibit the effect of the polypeptide on tissue cells istested. Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies, the preparation of whichwill be described hereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies:A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

G. Cell-Based Assays

Cell-based assays and animal models for immune related diseases can beused to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

In a different approach, cells of a cell type known to be involved in aparticular immune related disease are transfected with the cDNAsdescribed herein, and the ability of these cDNAs to stimulate or inhibitimmune function is analyzed. Suitable cells can be transfected with thedesired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are more commonly used in the art. Techniques toderive continuous cell lines from transgenic animals are well known inthe art (see, e.g., Small et al., Mol. Cell. Biol. 5: 642-648 [1985]).

One suitable cell based assay is the mixed lymphocyte reaction (MLR).Current Protocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes ofHealth, Published by John Wiley & Sons, Inc. In this assay, the abilityof a test compound to stimulate or inhibit the proliferation ofactivated T cells is assayed. A suspension of responder T cells iscultured with allogeneic stimulator cells and the proliferation of Tcells is measured by uptake of tritiated thymidine. This assay is ageneral measure of T cell reactivity. Since the majority of T cellsrespond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, above, 3.15, 6.3.

A proliferative T cell response in an MLR assay may be due to directmitogenic properties of an assayed molecule or to external antigeninduced activation. Additional verification of the T cell stimulatoryactivity of the polypeptide can be obtained by a costimulation assay. Tcell activation requires an antigen specific signal mediated through theT-cell receptor (TCR) and a costimulatory signal mediated through asecond ligand binding interaction, for example, the B7 (CD80, CD86)/CD28binding interaction. CD28 crosslinking increases lymphokine secretion byactivated T cells. T cell activation has both negative and positivecontrols through the binding of ligands which have a negative orpositive effect. CD28 and CTLA-4 are related glycoproteins in the Igsuperfamily which bind to B7. CD28 binding to B7 has a positivecostimulation effect of T cell activation; conversely, CTLA-4 binding toB7 has a T cell deactivating effect. Chambers, C. A. and Allison, J. P.,Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H., Cell (1992) 71:1065;Linsey, P. S, and Ledbetter, J. A., Annu. Rev. Immunol. (1993) 11:191;June, C. H. et al, Immunol. Today (1994) 15:321; Jenkins, M. K.,Immunity (1994) 1:405. In a costimulation assay, the polypeptides areassayed for T cell costimulatory or inhibitory activity.

Direct use of a stimulating compound as in the invention has beenvalidated in experiments with 4-1BB glycoprotein, a member of the tumornecrosis factor receptor family, which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol. (1994) 24:2219.

The use of an agonist stimulating compound has also been validatedexperimentally. As one example, activation of 4-1BB by treatment with anagonist anti-4-1BB antibody enhances eradication of tumors. Hellstrom,I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998) 18:1. Immunoadjuvanttherapy for treatment of tumors, described in more detail below, isanother example of the use of the stimulating compounds of theinvention.

Alternatively, an immune stimulating or enhancing effect can also beachieved by administration of a polypeptide which has vascularpermeability enhancing properties. Enhanced vascular permeability wouldbe beneficial to disorders which can be attenuated by local infiltrationof immune cells (e.g., monocytes, eosinophils, PMNs) and inflammation.

On the other hand, TIGIT polypeptides, as well as other compounds of theinvention, which are direct inhibitors of T cellproliferation/activation, proinflammatory cytokine secretion, and/orvascular permeability can be directly used to suppress the immuneresponse. These compounds are useful to reduce the degree of the immuneresponse and to treat immune related diseases characterized by ahyperactive, superoptimal, or autoimmune response. This use of thecompounds of the invention has been validated by the experimentsdescribed above in which CTLA-4 binding to receptor B7 deactivates Tcells. The direct inhibitory compounds of the invention function in ananalogous manner. The use of a compound which suppresses vascularpermeability would be expected to reduce inflammation. Such uses wouldbe beneficial in treating conditions associated with excessiveinflammation.

Similarly, compounds, e.g., antibodies, which bind to TIGIT-inhibitorypolypeptides and block the effect of these TIGIT-inhibitory polypeptidesproduce a net inhibitory effect and can also be used to suppress the Tcell mediated immune response by leaving TIGIT free to inhibit T cellproliferation/activation and/or lymphokine secretion. Blocking theinhibitory effect of the polypeptides suppresses the immune response ofthe mammal.

Alternatively, for conditions associated with insufficient T cellmediated immune response and/or inflammation, inhibiting or lesseningTIGIT activity and/or expression or interfering with TIGIT's ability tobind to and/or signal through PVR may be beneficial for treatment. Suchinhibition or lessening may be provided by administration of anantagonist of TIGIT expression and/or activity and/or an antagonist ofPVR expression and/or activity.

H. Animal Models

The results of the cell based in vitro assays can be further verifiedusing in vivo animal models and assays for T-cell function. A variety ofwell known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis ofimmune related disease, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them predictive of responses in humanpatients. Animal models of immune related diseases include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing cells into syngeneic mice usingstandard techniques, e.g., subcutaneous injection, tail vein injection,spleen implantation, intraperitoneal implantation, implantation underthe renal capsule, etc.

Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction and a measureof their role in transplant rejection. The most common and acceptedmodels use murine tail-skin grafts. Repeated experiments have shown thatskin allograft rejection is mediated by T cells, helper T cells andkiller-effector T cells, and not antibodies. Auchincloss, H. Jr. andSachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., RavenPress, NY, 1989, 889-992. A suitable procedure is described in detail inCurrent Protocols in Immunology, above, unit 4.4. Other transplantrejection models which can be used to test the compounds of theinvention are the allogeneic heart transplant models described byTanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al,J. Immunol. (1994) 4330-4338.

Animal models for delayed type hypersensitivity provides an assay ofcell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell andmononuclear cell inflammation and subsequent demyelination of axons inthe central nervous system. EAE is generally considered to be a relevantanimal model for MS in humans. Bolton, C., Multiple Sclerosis (1995)1:143. Both acute and relapsing-remitting models have been developed.The compounds of the invention can be tested for T cell stimulatory orinhibitory activity against immune mediated demyelinating disease usingthe protocol described in Current Protocols in Immunology, above, units15.1 and 15.2. See also the models for myelin disease in whicholigodendrocytes or Schwann cells are grafted into the central nervoussystem as described in Duncan, I. D. et al, Molec. Med. Today (1997)554-561.

Contact hypersensitivity is a simple delayed type hypersensitivity invivo assay of cell mediated immune function. In this procedure,cutaneous exposure to exogenous haptens which gives rise to a delayedtype hypersensitivity reaction which is measured and quantitated.Contact sensitivity involves an initial sensitizing phase followed by anelicitation phase. The elicitation phase occurs when the T lymphocytesencounter an antigen to which they have had previous contact. Swellingand inflammation occur, making this an excellent model of human allergiccontact dermatitis. A suitable procedure is described in detail inCurrent Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc.,1994, unit 4.2. See also Grabbe, S, and Schwarz, T, Immun. Today 19 (1):37-44 (1998).

An animal model for arthritis is collagen-induced arthritis. This modelshares clinical, histological and immunological characteristics of humanautoimmune rheumatoid arthritis and is an acceptable model for humanautoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996)88:569.

The collagen-induced arthritis (CIA) model is considered a suitablemodel for studying potential drugs or biologics active in humanarthritis because of the many immunological and pathologicalsimilarities to human rheumatoid arthritis (RA), the involvement oflocalized major histocompatibility, complete class-II-restricted Thelper lymphocyte activation, and the similarity of histologicallesions. Features of this CIA model that are similar to that found in RApatients include: erosion of cartilage and bone at joint margins (as canbe seen in radiographs), proliferative synovitis, symmetricalinvolvement of small and medium-sized peripheral joints in theappendicular, but not the axial, skeleton. Jamieson et al., Invest.Radiol. 20: 324-9 (1985). Furthermore, IL-1 and TN-α appear to beinvolved in CIA as in RA. Joosten et al., J. Immunol. 163: 5049-5055(1999). TNF-neutralizing antibodies and separately, TNFR:Fc reduced thesymptoms of RA in this model (Williams et al., PNAS, 89:9784-9788(1992); Wooley et al., J. Immunol. 151: 6602-6607 (1993)).

In this model for RA, type II collagen is purified from bovine articularcartilage (Miller, Biochemistry 11:4903 (1972)) and used to immunizedmice (Williams et al, Proc. Natl. Acad. Sci. USA 91:2762 (1994)).Symptoms of arthritis include erythema and/or swelling of the limbs aswell as erosions or defects in cartilage and bone as determined byhistology. This widely used model is also described, for example, byHolmdahl et al., APMIS 97:575 (1989) and in Current Protocols inImmunology, supra, units 15.5, and in Issekutz et al., Immunology,88:569 (1996), as well as in the Examples hereinbelow.

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia and inflammation are induced bysensitizing an animal with ovalbumin and then challenging the animalwith the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al, Am. J.Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.

Additionally, the compounds of the invention can be tested on animalmodels for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al, Nat. Med.(1997) 3:183, in which the mice demonstrate histopathologic skin lesionsresembling psoriasis. Another suitable model is the human skin/scidmouse chimera prepared as described by Nickoloff, B. J. et al, Am. J.Path. (1995) 146:580.

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g., baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell 56,313-321 [1989]); electroporation of embryos (Lo, Mol. Cel. Biol. 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell57, 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA 89, 6232-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to a polypeptide of theinvention, prepared as described above, are administered to the animaland the effect on immune function is determined.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a polypeptide identified herein, as aresult of homologous recombination between the endogenous gene encodingthe polypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

I. ImmunoAdjuvant Therapy

In one embodiment, the immunostimulating compounds of the invention canbe used in immunoadjuvant therapy for the treatment of tumors (cancer).It is now well established that T cells recognize human tumor specificantigens. One group of tumor antigens, encoded by the MAGE, BAGE andGAGE families of genes, are silent in all adult normal tissues, but areexpressed in significant amounts in tumors, such as melanomas, lungtumors, head and neck tumors, and bladder carcinomas. DeSmet, C. et al.,(1996) Proc. Natl. Acad. Sci. USA, 93:7149. It has been shown thatcostimulation of T cells induces tumor regression and an antitumorresponse both in vitro and in vivo. Melero, I. et al., Nature Medicine(1997) 3:682; Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA (1997) 94:8099; Lynch, D. H. et al, Nature Medicine (1997) 3:625; Finn, O. J. andLotze, M. T., J. Immunol. (1998) 21:114. The data provided hereindemonstrates that TIGIT expression correlates with immune cellinfiltrate in breast cancer tumors. TIGIT is also demonstrated herein toinhibit proliferation of DC and other immune cells and to inhibitproinflammatory cytokine production from such cells. Thus, TIGIToverexpression in tumor immune infiltrate cells may be aberrant, sincedecreased T cell activity in tumors would be undesirable. TIGITantagonists and/or antagonists of the TIGIT-PVR signaling interaction(i.e., PVR antagonists) may be administered as adjuvants, alone ortogether with a growth regulating agent, cytotoxic agent orchemotherapeutic agent, to stimulate T cell proliferation/activation andan antitumor response to tumor antigens. The growth regulating,cytotoxic, or chemotherapeutic agent may be administered in conventionalamounts using known administration regimes. Immunostimulating activityby the TIGIT-antagonistic and TIGIT activity-antagonistic compounds ofthe invention allows reduced amounts of the growth regulating,cytotoxic, or chemotherapeutic agents thereby potentially lowering thetoxicity to the patient.

J. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind to or complex with the polypeptides encoded by the genesidentified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art. All assays are commonin that they call for contacting the drug candidate with a polypeptideencoded by a nucleic acid identified herein under conditions and for atime sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g., on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

In order to find compounds that interfere with the interaction of a geneidentified herein and other intra- or extracellular components can betested, a reaction mixture is usually prepared containing the product ofthe gene and the intra- or extracellular component under conditions andfor a time allowing for the interaction and binding of the two products.To test the ability of a test compound to inhibit binding, the reactionis run in the absence and in the presence of the test compound. Inaddition, a placebo may be added to a third reaction mixture, to serveas positive control. The binding (complex formation) between the testcompound and the intra- or extracellular component present in themixture is monitored as described above. The formation of a complex inthe control reaction(s) but not in the reaction mixture containing thetest compound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

K. Compositions and Methods for the Treatment of Immune Related Diseases

The compositions useful in the treatment of immune related diseasesinclude, without limitation, proteins, antibodies, small organicmolecules, peptides, phosphopeptides, antisense and ribozyme molecules,triple helix molecules, etc. that inhibit or stimulate immune function,for example, T cell proliferation/activation, lymphokine release, orimmune cell infiltration.

For example, antisense RNA and RNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology 4, 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

L. Anti-TIGIT Antibodies

The present invention further provides anti-TIGIT antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies. It will be understood by one of ordinaryskill in the art that the invention also provides antibodies againstother polypeptides (i.e., anti-PVR antibodies) and that any of thedescription herein drawn specifically to the method of creation,production, varieties, use or other aspects of anti-TIGIT antibodieswill also be applicable to antibodies specific for other non-TIGITpolypeptides.

1. Polyclonal Antibodies

The anti-TIGIT antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the TIGIT polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-TIGIT antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the TIGIT polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-TIGIT antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forTIGIT, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. As onenonlimiting example, trispecific antibodies can be prepared. See, e.g.,Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven TIGIT polypeptide herein. Alternatively, an anti-TIGIT polypeptidearm may be combined with an arm which binds to a triggering molecule ona leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular TIGIT polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular TIGIT polypeptide. These antibodies possess a TIGIT-bindingarm and an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the TIGIT polypeptide and further binds tissue factor(TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising an antibodyconjugated to one or more cytotoxic agents, such as a chemotherapeuticagent, a drug, a growth inhibitory agent, a toxin (e.g., a proteintoxin, an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Immunoconjugates have been used for the local delivery of cytotoxicagents, i.e., drugs that kill or inhibit the growth or proliferation ofcells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion inPharmacology 5:543-549; Wu et al (2005) Nature Biotechnology23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos(1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer(1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278).Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and intracellular accumulation therein, where systemicadministration of unconjugated drugs may result in unacceptable levelsof toxicity to normal cells as well as the tumor cells sought to beeliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications (A. Pinchera et al., eds) pp. 475-506. Both polyclonalantibodies and monoclonal antibodies have been reported as useful inthese strategies (Rowland et al., (1986) Cancer Immunol. Immunother.21:183-87). Drugs used in these methods include daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins usedin antibody-toxin conjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may exerttheir cytotoxic effects by mechanisms including tubulin binding, DNAbinding, or topoisomerase inhibition. Some cytotoxic drugs tend to beinactive or less active when conjugated to large antibodies or proteinreceptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a huCD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5585089; 5606040;5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen,Inc.), an antibody-drug conjugate composed of the huC242 antibody linkedvia the disulfide linker SPP to the maytansinoid drug moiety, DM1, isadvancing into Phase II trials for the treatment of cancers that expressCanAg, such as colon, pancreatic, gastric, and other cancers. MLN-2704(Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drugconjugate composed of the anti-prostate specific membrane antigen (PSMA)monoclonal antibody linked to the maytansinoid drug moiety, DM1, isunder development for the potential treatment of prostate tumors. Theauristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE),synthetic analogs of dolastatin, were conjugated to chimeric monoclonalantibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specificto CD30 on hematological malignancies) (Doronina et al (2003) NatureBiotechnol. 21(7):778-784) and are under therapeutic development.

In certain embodiments, an immunoconjugate comprises an antibody and achemotherapeutic agent or other toxin. Chemotherapeutic agents useful inthe generation of immunoconjugates are described herein (e.g., above).Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

a. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

b. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lake, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

c. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ1I,α2I, α3I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

d. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc99m or 1123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc⁹⁹m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A.). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

e. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g. about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC’), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither glactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

M. Pharmaceutical Compositions

The active molecules of the invention (e.g., TIGIT polypeptides,anti-TIGIT antibodies, variants of each, TIGIT agonists, TIGITantagonists, PVR agonists and PVR antagonists) as well as othermolecules identified by the screening assays disclosed above, can beadministered for the treatment of immune related diseases, in the formof pharmaceutical compositions.

Therapeutic formulations of an active molecule, for example apolypeptide or antibody of the invention, are prepared for storage bymixing the active molecule having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compounds identified by the screening assays disclosed herein can beformulated in an analogous manner, using standard techniques well knownin the art.

Lipofections or liposomes can also be used to deliver the activemolecule into cells. Where antibody fragments are used, the smallestinhibitory fragment which specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variableregion sequences of an antibody, peptide molecules can be designed whichretain the ability to bind the target protein sequence. Such peptidescan be synthesized chemically and/or produced by recombinant DNAtechnology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90,7889-7893 [1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active molecules may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations of the active molecules may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

N. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat various immunerelated diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of inflammatory cells intoa tissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased cytokine production, and/orincreased or decreased vascular permeability or the inhibition thereof.Given the disclosures herein of TIGIT's role in modulating T cellproliferation and cytokine production, modulation of TIGIT expressionand/or activity may be efficacious in preventing and/or treating thesediseases.

Exemplary conditions or disorders to be treated with the polypeptides,antibodies and other compounds of the invention, include, but are notlimited to systemic lupus erythematosis, rheumatoid arthritis, juvenilechronic arthritis, osteoarthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. Antibodies eitherdirectly or indirectly mediate tissue injury. Though T lymphocytes havenot been shown to be directly involved in tissue damage, T lymphocytesare required for the development of auto-reactive antibodies. Thegenesis of the disease is thus T lymphocyte dependent. Multiple organsand systems are affected clinically including kidney, lung,musculoskeletal system, mucocutaneous, eye, central nervous system,cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self IgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid if infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, intestitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrhematoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rhematoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing sponylitis, Reiter's syndrome(reactive arthritis), arthritis associated with inflammatory boweldisease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8⁺ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8⁺ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class 1molecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8⁺T cells response. As shown herein, TIGIT is expressed in CD8⁺ T cells,and modulation of TIGIT expression and/or activity in those cells maymodulate the symptoms of and/or prevent this disease.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin; likely this is induced by anactive inflammatory process. Scleroderma can be localized or systemic;vascular lesions are common and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

Sjögren's syndrome is due to immune-mediated inflammation and subsequentfunctional destruction of the tear glands and salivary glands. Thedisease can be associated with or accompanied by inflammatory connectivetissue diseases. The disease is associated with autoantibody productionagainst Ro and La antigens, both of which are small RNA-proteincomplexes. Lesions result in keratoconjunctivitis sicca, xerostomia,with other manifestations or associations including bilary cirrhosis,peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet β cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including Multiple Sclerosis; idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome; and Chronic Inflammatory DemyelinatingPolyneuropathy, are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. In MS there is evidence to suggest that diseaseinduction and progression is dependent on T lymphocytes. MultipleSclerosis is a demyelinating disease that is T lymphocyte-dependent andhas either a relapsing-remitting course or a chronic progressive course.The etiology is unknown; however, viral infections, geneticpredisposition, environment, and autoimmunity all contribute. Lesionscontain infiltrates of predominantly T lymphocyte mediated, microglialcells and infiltrating macrophages; CD4+ T lymphocytes are thepredominant cell type at lesions. The mechanism of oligodendrocyte celldeath and subsequent demyelination is not known but is likely Tlymphocyte driven.

Inflammatory and Fibrotic Lung Disease, including EosinophilicPneumonias; Idiopathic Pulmonary Fibrosis, and HypersensitivityPneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

Autoimmune or Immune-mediated Skin Disease including Bullous SkinDiseases, Erythema Multiforme, and Contact Dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

Transplantation associated diseases, including Graft rejection andGraft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Other diseases in which intervention of the immune and/or inflammatoryresponse have benefit are infectious disease including but not limitedto viral infection (including but not limited to AIDS, hepatitis A, B,C, D, E and herpes) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.

It has been demonstrated that some human cancer patients develop anantibody and/or T lymphocyte response to antigens on neoplastic cells.It has also been shown in animal models of neoplasia that enhancement ofthe immune response can result in rejection or regression of thatparticular neoplasm. Molecules that enhance the T lymphocyte response inthe MLR have utility in vivo in enhancing the immune response againstneoplasia. Molecules which enhance the T lymphocyte proliferativeresponse in the MLR (or small molecule agonists or antibodies thataffected the same receptor in an agonistic fashion) can be usedtherapeutically to treat cancer. Molecules that inhibit the lymphocyteresponse in the MLR (i.e., TIGIT) also function in vivo during neoplasiato suppress the immune response to a neoplasm; such molecules can eitherbe expressed by the neoplastic cells themselves or their expression canbe induced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

Additionally, inhibition of molecules with proinflammatory propertiesmay have therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatis.

The compounds of the present invention, e.g., polypeptides, smallmolecules or antibodies, are administered to a mammal, preferably ahuman, in accord with known methods, such as intravenous administrationas a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation (intranasal, intrapulmonary) routes. Intravenous,subcutaneous or inhaled administration of polypeptides and antibodiesare most commonly used.

In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with, e.g., animmunoadjuvant of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-estrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

It may be desirable to also administer antibodies against other immunedisease associated or tumor associated antigens, including, but notlimited to antibodies which bind to CD20, CD11a, CD18, ErbB2, EGFR,ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, orin addition, two or more antibodies binding the same or two or moredifferent antigens disclosed herein may be coadministered to thepatient. Sometimes, it may be beneficial to also administer one or morecytokines to the patient. For example, in one embodiment, the TIGITpolypeptides are coadministered with a growth inhibitory agent. Forexample, the growth inhibitory agent may be administered first, followedby a TIGIT polypeptide. However, simultaneous administration oradministration first is also contemplated. Suitable dosages for thegrowth inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth inhibitory agent and the,e.g., TIGIT polypeptide.

For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound may be suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

O. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials (e.g., comprising a TIGIT molecule, TIGIT agonist,TIGIT antagonist, PVR agonist, or PVR antagonist) useful for thediagnosis or treatment of the disorders described above is provided. Thearticle of manufacture comprises a container and an instruction.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for diagnosing or treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide or an antibody of the invention. An instruction or label on,or associated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

P. Diagnosis and Prognosis of Immune Related Disease

Cell surface proteins, such as proteins which are overexpressed incertain immune related diseases (i.e., TIGIT), are excellent modulationtargets for drug candidates or disease treatment. The same proteinsalong with secreted proteins encoded by the genes amplified in immunerelated disease states find additional use in the diagnosis andprognosis of these diseases. For example, antibodies directed againstthe protein products of genes amplified in rheumatoid arthritis or otherimmune related diseases can be used as diagnostics or prognostics.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by amplified or overexpressed genes (“marker gene products”).The antibody preferably is equipped with a detectable, e.g., fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the overexpressed gene encodes a cell surfaceprotein Such binding assays are performed essentially as describedabove.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection. Other techniques are also well known in the art, forexample fluorescence-assisted cell sorting (FACS).

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent, patent publication and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

EXAMPLES Example 1 Further Characterization of TIGIT

TIGIT had been previously identified (see, e.g., US patent publicationno. US20040121370, incorporated herein by reference in its entirety) ingenome-wide search strategies targeting genes specifically expressed byimmune cells which have a domain structure consisting of extracellularIg domains, a type one transmembrane region, and an intracellularimmunoreceptor tyrosine-based activation or inhibition (ITAM/ITIM)motif(s) (Abbas, A. R. et al. Genes Immun 6, 319-31 (2005); Burshtyn, D.N. et al., J Biol Chem 272, 13066-72 (1997); Kashiwada, M. et al., JImmunol 167, 6382-7 (2001)). The sequence of human TIGIT and homologuesfrom mouse (submitted to Genbank), rhesus monkey (Genbank accession no.XP_(—)001107698) and dog (Genbank accession no. XP_(—)545108) are shownin FIG. 1. To further elucidate the role of TIGIT in immune function, ahomology search was performed which identified the TIGIT Ig domain asbeing similar to the N-terminal IgV domains of the poliovirus receptor(PVR) protein and PVR-like proteins 1-4 (PVRL1-4), as well as theN-terminal IgV domains of CD96 and CD226 (see FIGS. 2A-2B). Thealignment of these proteins showed that the highly conserved residuesthat define the canonical IgV domain were conserved in TIGIT, andfurther suggested that those eight proteins may comprise a relatedsubset of the Ig family. The conserved V-frame residues have been shownto be important for establishing the V-frame fold (Wiesmann, C. & deVos, A. M. Cell Mol Life Sci 58, 748-59 (2001)). A number of residueswere identified near the V-frame fold that were conserved among theeight proteins, including four absolutely conserved residues (A⁶⁷, G⁷⁴,P¹¹⁴, and G¹¹⁶) and five conserved residues (V/I/L⁵⁴, S/T⁵⁵, Q⁵⁶, T¹¹²and F/Y¹¹³) that comprise three submotifs (V/I⁵⁴-S/T⁵⁵-Q⁵⁶),(A⁶⁷-X(6)-G⁷⁴) and (T¹¹²-F/Y¹¹³-P¹¹⁴-X-G¹¹⁶.) In the case of TIGIT,these submotifs appear to be conserved across species (see FIG. 1) andare not present in other currently described IgV domain containingproteins. Those conserved residues may define a class of PVR-likeproteins including PVR, PVR-like proteins 1-4, CD96, CD226, and TIGIT.

PVRL1-4 and PVR share a common domain architecture (IgV-IgC-IgV),whereas CD226 and CD96 lack the membrane proximal IgV domain. TIGIT isthe most economical member of the family, consisting of a single IgVdomain. The intracellular segments of these eight proteins show only alimited similarity with each other outside of the afadin binding motifshared between PVRL1-3. Based on the crystal structure of the relatedIgV domain of NECL-1 (Dong, X. et al., J Biol Chem 281, 10610-7 (2006)),the first and third motifs are predicted to lie in hairpin loops betweenthe B and C and the F and G beta-strands, respectively. These two loopsare adjacent to each other at one end o the IgV fold. The second motifcomprises the C′ and C″ beta strands that are involved in forming partof the homodimeric interface for NECL-1. Thus, the observed sequencemotifs in the TIGIT/PVR family may play a role in specific homo- andheterotypic interactions observed between PVR family members. PVR haspreviously been characterized as a nectin-like protein, but the abovesequence analysis suggests that it should instead be considered a PVRfamily member, with certain nectins (i.e., PVRL1-4) being categorized asa branch of the PVR family.

Example 2 Identification of PVR Ligand

Potential binding partners for TIGIT were identified by screening alarge library of secreted proteins to look for proteins that boundimmobilized TIGIT. Briefly, an Fc fusion of TIGIT (TIGIT-Fc) wasconstructed by cloning amino acids 1-138 of human TIGIT into a vectorimmediately preceding the Fc region of human IgG1 (TIGIT-Fc). Analternate version of TIGIT-Fc in which FcγR binding was abolished wasalso constructed by introducing two mutations into the Fc tail ofTIGIT-Fc at D256A and N297A using standard site-directed mutagenesistechniques (TIGIT-Fc-DANA). The resulting fusion protein was transientlyexpressed in and purified from CHO cells using standard affinitychromatography techniques. A library of individual secreted proteinsfused to hexahistidine or Fc tags were screened for binding to TIGIT-Fcusing the Octet system (ForteBio). Proteins were tested for binding inHBS-P (10 mM Hepes, pH 7.4; 0.15M NaCl; 0.005% Surfactant P20). TIGIT-Fcor a control Fc fusion protein was loaded onto anti-human Fc biosensorsto saturation. The biosensors were washed in buffer (30 seconds), placedinto wells containing 5 μg/mL protein for three minutes, and washedagain for 30 seconds. The sensors were reloaded and washed after everytwo binding cycles. Binding was indicated as an increase in responselevel greater than 0.2 nm, and specificity was determined by comparisonto a control Fc fusion protein. A single protein that bound TIGIT wasidentified in over 1000 proteins analyzed. As shown in FIG. 3, aTIGIT-Fc fusion protein immobilized onto an anti-human Fc biosensorspecifically interacted with a PVR-Fc fusion protein. The specificity ofthis interaction was supported by the lack of specific interaction ofTIGIT with any other protein in the library, and further by the factthat biosensors loaded with other Ig domain-containing proteins did notelicit a response to PVR.

Because it had previously been known that PVR, PVRL1-4, CD96, and CD226interact with one another (He, Y. et al., J Virol 77, 4827-35 (2003);Satoh-Horikawa, K. et al., J Biol Chem 275, 10291-9 (2000); Bottino, C.et al. J Exp Med 198, 557-67 (2003); Fuchs, A. et al., J Immunol 172,3994-8 (2004); Reymond, N. et al., J Exp Med 199, 1331-41 (2004)), theinteraction of TIGIT with each of these proteins was assessed using thebiosensor system described above. Fc fusion proteins were constructedand purified for each of the proteins to be tested as described abovefor TIGIT-Fc. Specifically, amino acids 1-343 of PVR-like protein 1(PVRL1), amino acids 1-360 of PVR-like protein 2 (PVRL2), amino acids1-411 of PVR-like protein 3 (PVRL3), amino acids 1-349 of PVR-likeprotein 4 (PVRL4), amino acids 1-259 of CD226, or amino acids 1-500 ofCD96 were fused immediately preceding the Fc region of human IgG1. Theresulting Fc fusion proteins were tested for binding to TIGIT-Fc.PVR-Fc, PVRL3-Fc and PVRL2-Fc bound TIGIT-Fc, whereas CD226-Fc, CD96-Fc,PVRL1-Fc, and PVRL4-Fc did not bind TIGIT-Fc (FIG. 4A). Of the threeobserved binders, PVR-Fc showed the greatest binding to TIGIT-Fc,followed by PVRL3-Fc, and the least amount of binding of the three toTIGIT-Fc was observed with PVRL2-Fc.

FACS analyses were also performed to assess the binding of the PVRfamily member Fc fusions constructed above to CHO cells expressingTIGIT. Fc fusion proteins were biotinylated via amine coupling usingNHS-PEO4-Biotin (Pierce) in PBS. Binding of biotin-ligands was detectedusing phycoerythrin-conjugated streptavidin (Caltag). Mouse monoclonalantibody to gD tag (Genentech) was conjugated to AlexaFluor 647(Invitrogen). Antibodies were conjugated to appropriate fluor labelsusing standard techniques. Cells were stained per the manufacturer'sinstructions. Prior to staining, cells were blocked with appropriatesera or purified IgG. Acquisition was performed on a FACSCalibur (BDBiosciences) and analyzed with JoFlo software (Tree Star, Inc.). Forwardand side scatter gated viable cells. The results are set forth in FIGS.4B-1 to 4B-6, and show that the binding pattern observed in theartificial biosensor assay was the same as that observed in a morephysiological setting at the cell surface.

To determine the strength of the PVR-TIGIT, PVRL2-TIGIT and PVRL3-TIGITbinding interactions, direct radioligand binding assays were performedusing CHO cells stably transfected with those proteins. For CHO cellsurface expression, TIGIT, PVR, PVRL2, PVRL3, CD226 and CD96 full-lengthDNAs were cloned into a vector immediately following a gD signalsequence (MGGTAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 19)) and the gD tag(KYALADASLKMADPNRFRGKDLPVL (SEQ ID NO: 20)). Plasmids were transfectedinto CHO cells using Lipofectamine LTX (Invitrogen). Expression ofgD-tagged proteins was verified by flow cytometry using the Alexa-647anti-gD conjugate. Stably-transfected cell lines were sorted twice byFACS for purity before use. Fc-fusion proteins constructed as describedabove were iodinated (¹²⁵I) using the Iodogen method. Binding studieswere carried out on stable transfectants in triplicate with 0.1-3 nMiodinated ligand. Iodinated proteins were incubated with 1×10⁵-2×10⁵cells in the presence of a dilution series of unlabeled competitorprotein (25 pM-5 μM) for four hours at 4° C. Cell suspensions wereharvested onto nitrocellulose membranes (Millipore) and washedextensively. Dried filters were counted and Scatchard analyses wereperformed using NewLigand 1.05 software (Genentech) to determine bindingaffinity (K_(d)).

FIGS. 5A and 5B show the binding of the radiolabeled TIGIT-Fc protein toPVR-expressing CHO cells. The average Kd for the TIGIT-Fc-PVRinteraction over four experiments was 3.15 nM. Table 6 shows the resultsof all the analyses in tabular form.

TABLE 6 Cell binding of PVR family proteins. Receptors were expressed onCHO cells, and all ligands were-Fc constructs. MFI was determined byflow cytometry with biotinylated Fc- ligands, after gating onreceptor-positive cells. Binding affinity (Kd) was determined bycompetition radioligand binding assay. Kd is indicated (nM) and is theaverage value from at least 3 independent assays, except where indicated(*). Ligand PVR TIGIT PVRL2 PVRL3 CD226 CD96 Receptor MFI Kd MFI Kd MFIKd MFI Kd MFI Kd MFI PVR − − +++ 1.02 − − +++ 70.8 +++ 114* +++ TIGIT++++ 3.15 − − ++ & +++ 38.9 − − − PVRL2 − − − − − − ++ 14-30 − − − PVRL3++ − ++ − +++ 3-13 − − − − − CD226 +++ 119 − − + & − − − − − CD96 +++37.6 − − − − − − − − − ++++ MFI > 5000 +++ MFI = 1000-4999 ++ MFI =100-999 + MFI < 100 − No binding & Specific binding but Kd notelucidated *average of two assaysThe interaction of TIGIT with PVR exhibited the highest affinity (Kd=1-3nM) while the affinity of TIGIT binding to PVRL3 was approximately10-30-fold lower (Kd=38.9 nM) (see Table 6). Due to poor curve fittingin the radioligand assay the binding constant for the PVRL2-TIGITinteraction could not be determined, but specific binding wasnonetheless observed and was consistent with the above-described FACSdata showing modest binding of PVRL2-Fc to CHO-TIGIT, and furtherbolstered the finding that binding between PVRL2 and TIGIT is alow-affinity interaction. Iodinated Fc fusion protein (ligand) was boundto receptor-expressing CHO cells at the indicated concentration, andcompeted with 10-fold serial dilutions of CD226-Fc (8 μM on CHO-TIGIT; 5μM on CHO-PVR), TIGIT-Fc (2 μM on CHO-PVR; 6 μM on CHO-CD226 andCHO-CD96). Non-specific binding was determined using 2000-fold excesscold ligand and subtracted from total binding. The competition studiesshowed that TIGIT effectively blocked the interaction of PVR to itsother co-receptors CD226 and CD96, whereas CD226 was a less effectiveinhibitor of the TIGIT-PVR interaction (FIG. 6). This data was inagreement with the higher observed affinity of the PVR-TIGIT interaction(1-3 nM) as compared to the PVR-CD226 interaction (approximately 115 nM,according to Tahara-Hanaoka, S. et al. Int Immunol 16, 533-8 (2004)).Direct competition studies with CD96 were not possible due to lowexpression of that protein, although TIGIT completely inhibited PVRbinding to CD96-expressing CHO cells. The foregoing competition studiesdemonstrated that TIGIT, CD226, and CD96 share a common binding site oroverlapping binding sites on PVR. This finding was further supported bythe observation that the anti-PVR antibody D171, which binds to theN-terminal IgV domain of PVR, blocks the binding of TIGIT and CD226 toPVR (FIG. 7).

Example 3 Expression of TIGIT and PVR

(A) Expression of TIGIT and PVR on Resting and Activated Immune Cells

The relative distribution and expression of TIGIT and PVR on immunecells was assessed as an indicator of the role of these two molecules innormal immune function, and was compared to the expression of CD226, amolecule known previously and shown in Example 2 to interact with PVR invivo. An earlier study had shown that the expression of TIGIT wasspecific to T and NK cells, across multiple immune cell types as well asan array of tissues (Abbas, A. R. et al., Genes Immun 6, 319-31 (2005)).A further analysis of the expression of TIGIT in a variety of immunecells and tissues ex vivo and after activation was performed. As shownin FIGS. 8A and 8B, TIGIT is most strongly expressed in regulatory Tcells (T_(reg)), and is also highly expressed in NK cells and T_(fh)cells from human tonsillar tissue. TIGIT is expressed to a lesser extentin unstimulated NK cells, in activated and resting memory T cells, inCD8⁺ T cells and in Th2 and Th1 cells. This data correlates with thedata shown in US patent publication no. US20040121370, where TIGIT wasshown to be significantly overexpressed in isolated CD4⁺ T cellsactivated by anti-CD3/ICAM-1 and anti-CD3/anti-CD28 as compared toisolated resting CD4⁺ T cells. By contrast, PVR has been reported to beexpressed in endothelial cells, fibroblasts, osteoclasts, folliculardendritic cells, dendritic cells, and tumor cells (Sakisaka, T. & Takai,Y., Curr Opin Cell Biol 16, 513-21 (2004); Fuchs, A. & Colonna, M.,Semin Cancer Biol 16, 359-66 (2006)). This data highlights that TIGIT isassociated with T cells that produce regulatory cytokines that maysuppress the immune response.

Complementary flow cytometric analyses were also performed, using thesame methods as described in Example 2. Human ex vivo T cells wereexamined after activation for surface TIGIT expression using a hamsteranti-murine TIGIT antibody (10A7) that cross-reacts to human TIGIT andblocks TIGIT interaction with PVR (see FIG. 9). Anti-TIGIT antibodieswere generated by immunizing hamsters with murine TIGIT-Fc fusionprotein and obtaining hamster-anti-mouse antibodies therefrom usingstandard techniques. Two antibodies, 10A7 and 1F4, also specificallybound to human TIGIT (data not shown) and were used for furtherexperiments. Notably, 10A7 and 1F4 bind to different epitopes on humanTIGIT, as evidenced by the fact that 1F4 binding to TIGIT does not block10A7 binding to TIGIT on the surface of 293 cells expressing TIGIT (datanot shown). The amino acid sequences of the light and heavy chains ofthe 10A7 antibody were determined using standard techniques. The lightchain sequence of this antibody is:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO: 21) and the heavy chain sequence of this antibody is:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO: 22), where the complementarity determining regions (CDRs) of eachchain are represented by bold text. Thus, CDR1 of the 10A7 light chainhas the sequence KSSQSLYYSGVKENLLA (SEQ ID NO: 23), CDR2 of the 10A7light chain has the sequence ASIRFT (SEQ ID NO: 24), and CDR3 of the10A7 light chain has the sequence QQGINNPLT (SEQ ID NO: 25). CDR1 of the10A7 heavy chain has the sequence GFTFSSFTMH (SEQ ID NO: 26), CDR2 ofthe 10A7 heavy chain has the sequence FIRSGSGIVFYADAVRG (SEQ ID NO: 27),and CDR3 of the 10A7 heavy chain has the sequence RPLGHNTFDS (SEQ ID NO:28).

The amino acid sequences of the light and heavy chains of the 1F4antibody were determined using 5′ RACE (see, e.g., Ozawa et al.,BioTechniques 40(4): 469-478 (2006)). The light chain sequence of thisantibody is:DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO: 29) andthe heavy chain sequence of this antibody is:EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO: 30), where the complementarity determining regions (CDRs) of eachchain are represented by bold text. Thus, CDR1 of the 1F4 light chainhas the sequence RSSQSLVNSYGNTFLS (SEQ ID NO: 31), CDR2 of the 1F4 lightchain has the sequence GISNRFS (SEQ ID NO: 32), and CDR3 of the 1F4light chain has the sequence LQGTHQPPT (SEQ ID NO: 33). CDR1 of the 1F4heavy chain has the sequence GYSFTGHLMN (SEQ ID NO: 34), CDR2 of the 1F4heavy chain has the sequence LIIPYNGGTSYNQKFKG (SEQ ID NO: 35), and CDR3of the 1F4 heavy chain has the sequence GLRGFYAMDY (SEQ ID NO: 36). Theprimers used for the RACE sequencing methodology were as follows: RT-PCRgene-specific primers: (i) heavy chain: IgGRace4:TTTYTTGTCCACCKTGGTGCTGC (SEQ ID NO: 37); IgGRace2:CTGGACAGGGATCCAGAGTTCC (SEQ ID NO: 38); IgGRace7: CARGTCAMDGTCACTGRCTCAG(SEQ ID NO: 39); IgGRace1: GAARTARCCCTTGACCAGGC (SEQ ID NO:64); (ii)light chain: KapRace3: GTAGAAGTTGTTCAAGAAG (SEQ ID NO: 40); KapRace2:GAGGCACCTCCAGATGTTAAC (SEQ ID NO: 41); KapRace7: CTGCTCACTGGATGGTGGGAAG(SEQ ID NO: 42); KapRace1: GAAGATGGATACAGTTGGTGC (SEQ ID NO: 43); and 5′RACE tail PCR primers: ODC2:GATTCAAATCTCAATTATATAATCCGAATATGTTTACCGGCTCGCTCATGGACCCCC CCCCCCCDN (SEQID NO: 44); ODC3: GAATTCCCCCCCCCCCCCC (SEQ ID NO: 45); ODC4:CTCATGGACCCCCCCCCCCC (SEQ ID NO: 46); ODC5: AAATATAATACCCCCCCCCCCCCC(SEQ ID NO: 47); ADCS: AAATATAATACCCCCCC (SEQ ID NO: 48), and ADC5X:CTCATGGACCCCCCC (SEQ ID NO: 49).

The nucleotide sequence encoding the 1F4 light chain was determined tobe GATGTTGTGTTGACTCAAACTCCACTCTCCCTGTCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGTAAACAGTTATGGGAACACCTTTTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCACAGCTCCTCATCTTTGGGATTTCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTACGCATCAGCCTCCCACGTTCGGTCCTGGGACCAAGCTGGAGGTGAAA (SEQ ID NO: 50) andthe nucleotide sequence encoding the 1F4 heavy chain was determined tobe GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAACTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCATCTTATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTATTCCTTACAATGGTGGTACAAGCTATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACTTCTGATGACTCTGCAGTCTATTTCTGTTCAAGAGGCCTTAGGGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 51).

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats by centrifugation over Ficoll-Paque Plus (Amersham Biosciences).Indicated subsets of cells were purified with corresponding MACS kits(Miltenyi). Purity of sorted cells was verified by flow cytometry andranged from greater than 93% for cells purified by magnetic cell sortingto greater than 98% for cells purified by flow cytometry. All cells wereblocked with 10-20% of the appropriate sera or purified IgG prior tostaining Quantitative PCR analyses were performed to assess the mRNAlevels of proteins of interest in the sorted cell populations. Total RNAof the sorted cells was isolated with an RNeasy kit (Qiagen) anddigested with DNAse I (Qiagen). Total cellular RNA wasreverse-transcribed and analyzed by real-time TaqMan™ PCR in triplicateaccording to the manufacturer's instructions using a 7500 SequenceDetection System (Applied Biosystems). Arbitrary expression units aregiven as fold-expression over unstimulated cells. The forward andreverse primers used to detect TIGIT were: TGCCAGGTTCCAGATTCCA (SEQ IDNO: 52) and ACGATGACTGCTGTGCAGATG (SEQ ID NO: 53), respectively, and theTIGIT probe sequence used was AGCCATGGCCGCGACGCT (SEQ ID NO: 54).

CD4⁺ T cells were isolated from PBMC and activated with anti-CD3 andanti-CD8. Cell surface-expressed TIGIT was undetectable in unstimulatednaïve CD4⁺CD45RA⁺ cells, whereas unstimulated CD4⁺CD45RO⁺ cells had lowbut detectable expression (FIGS. 10A-1 to 10A-2). As shown in FIGS.10A-1 to 10A-2, TIGIT expression differed significantly from CD226expression in RA⁺ vs. RO⁺ subsets of CD4 T cells. Analysis of mRNA inimmune cell populations sorted directly ex vivo from PBMC showed greaterexpression of TIGIT in T_(reg), RO, and NK cells than in other celltypes studied relative to TIGIT expression in naïve CD4⁺CD45RA⁺ cells(FIG. 10B). After activation with anti-CD3 and CD28, cellsurface-expressed TIGIT was upregulated in both naïve and memory T cellpopulations, as shown in FIGS. 10A-1 to 10A-2. CD4⁺CD45RO⁺ memory cellshad significantly higher levels of expression at 24 and 48 hourspost-activation as compared to CD4⁺CD45RA⁺ naïve cells (FIGS. 10A-1 to10A-2). The CD4⁺CD45RO⁺ memory cells expressed 5.3-fold more TIGIT mRNAat day 1 than on day 0, whereas naïve cells only increased expression ofTIGIT by 1.4-fold relative to day 0 (FIG. 10C). TIGIT expression was notdetectable by day 6.

The stability of TIGIT expression on T cells was also assessed. Briefly,CD4⁺CD45RO⁺ cells were isolated and activated with anti-CD3/anti-CD28for one day. The cells were flow sorted by FACS for CD4⁺ and CD4⁺TIGIT⁺populations. After resting for five days post-sorting, cells wererestimulated with anti-CD3/anti-CD28 for up to three days and the cellsurface TIGIT expression was determined by FACS. In a separateexperiment, sorted TIGIT⁺ cells and CD4⁺ cells were plated at a densityof 2×10⁵ cells/well onto 96-well plates coated with variousconcentrations of anti-CD3 (0-0.8 μg/mL), 100 μL volume and cultured for4 days under standard conditions. ³H-thymidine was added for the final18 hours of incubation, followed by washing. At the end of four days,the cells were solubilized and the radioactivity associated with eachsample was measured by scintillation counting. As shown in FIGS. 12A and12B, TIGIT expression was induced in both TIGIT⁺ cells and TIGIT⁻ cells,indicating that TIGIT⁻ cells can express TIGIT under certaincircumstances and that TIGIT⁺ cells are not a fixed cell population.

Given the higher level of TIGIT expression on effector memory cells,expression in T cell subsets was further dissected. Given thatco-stimulatory or co-inhibitory molecules expressed on activatedeffector/memory T cells are often expressed on induced T_(regs), TIGITexpression in T_(regs) was assessed. T_(regs) are phenotypically definedas CD25^(hi) cells, and are known to express the transcription factorFoxP3 (Fontenot, J. D. et al., Immunity 22, 329-41 (2005)). In mice, thetranscription factor FoxP3 is used to co-define T_(reg) populations(Linsley, P. S. et al., Science 257, 792-5 (1992)). However, thisassociation is not maintained in human T cells, since all activatedhuman T cells express FoxP3 (Ziegler S F., Eur J. Immunol. 37(1):21-3(2007))). Ex vivo freshly isolated CD4⁺CD25^(hi) cells expressed TIGIT,whereas CD25⁻ cells were negative for cell surface expression of TIGIT(Figure R(D)). TIGIT⁺ T cells also co-expressed FoxP3 and GITR (FIGS. 9and 10E). Activation of sorted CD25⁺ cells resulted in an upregulationof TIGIT protein expression (FIG. 10F) and a 6.5-fold increase in mRNAlevels (FIGS. 10C and 10F). The fold-increase in TIGIT mRNA wasequivalent in T_(reg) and memory T cells.

Comparison of mRNA levels from immune cells sorted directly ex vivo fromdonor PBMC showed that CD4⁺CD25^(hi) T_(regs), CD4⁺CD45RO⁺, and NK cellseach had significant TIGIT expression, with T_(regs) exhibiting thehighest expression (FIG. 10C). TIGIT expression was not observed inresting or activated B cells or monocytes (FIG. 10C and data not shown).Notably, CD226, another co-receptor for PVR, was not upregulated inCD4⁺CD25^(hi) T_(reg) cells, suggesting divergent regulatory roles ofTIGIT versus CD226 (FIG. 11).

In other experiments, TIGIT expression on human tonsil T (T_(FH)) cellswas examined using flow cytometry, following standard protocols asdescribed above, with the exception that for assays involving FoxP3,cells were stained with antibodies following the above protocol,followed by fixation and permeabilization of the cells and staining withanti-FoxP3 or control IgG. TIGIT expression correlated with high levelsof co-expression of CXCR5 and ICOS in T cells, markers which aretypically observed in T_(FH) cells (FIG. 8A). By contrast, CD226 (DNAM)expression in those cells was low to nonexistent (FIG. 8A). High levelsof TIGIT expression were also observed in CD4⁺CCR4⁺CCR6⁺ IL-17-producingTh cells (FIG. 14). Overall, TIGIT was shown to be expressed by restingand activated T regulatory cells, human tonsillar T_(fh) cells,IL17-producing helper T cells, resting and activated effector/memory Thelper cells (CD4⁺CD45RO⁺ cells) and NK cells, and can be furtherupregulated upon activation of these cells. CD8⁺ cells also expressTIGIT and this expression is only slightly upregulated upon cellularactivation. CD226 is shown herein and is known in the art to beexpressed by CD8⁺ T cells, on CD45RA⁺ T cells, mast cells, platelets,natural killer (NK) cells, activated CD4⁺CD45RA⁺ T cells, andCD4⁺CD45RO⁺ T cells. TIGIT is specifically expressed on T_(reg) andT_(Fh) and resting effector/memory CD4⁺CD45RO⁺ cells; whereas CD226 isnot expressed in these cells.

(B) Expression of TIGIT and PVR in Human Disease

Having determined that TIGIT is highly expressed on selected populationsof immune cells, the expression levels of TIGIT, PVR, and CD226 werenext assessed in tissues from different immune-related disease states,including psoriasis, inflammatory bowel disorder, arthritis, asthma, andcancer. A microarray-based system was used for the studies, anddescription of the appropriate microarray protocol can be found in theliterature, for example in US patent publication no. US20080038264,incorporated herein by reference. As shown in FIG. 15, significantexpression of TIGIT was observed in inflamed human synovial tissuerelative to uninflamed tissue, particularly notable in the case ofrheumatoid arthritis tissue. Within the inflamed arthritis tissuesamples, TIGIT expression was correlated mainly with T cells as opposedto macrophages or fibroblasts (see FIG. 15, right panel). This data wasfurther confirmed in murine collagen-induced arthritis (CIA) models byRT-PCR analysis of TIGIT mRNA levels (see FIG. 16). In the CIA modelused herein, DBA-1J mice were immunized with 100 μg bovine collagen typeII in 100 μL of Complete Freund's Adjuvant (CFA) on Day 0 and Day 21intradermally. RNA was extracted from joints from hind paws on days 28,30 and 40 and assessed for TIGIT and CD226 expression as describedabove. As seen in FIG. 16, increased TIGIT expression was observed atday 40, while CD226 expression was significantly downregulated by day40.

Lesser increases in expression of TIGIT relative to normal tissues wereobserved in psoriasis tissue samples and inflammatory bowel diseasetissue samples. Similar analyses in asthma tissue samples from rhesusmonkeys showed that TIGIT expression is significantly elevated indiseased tissue as compared to normal control tissue (FIG. 17). Breastcancer samples also exhibited greatly increased expression of TIGITrelative to normal breast tissue, with varying amounts in differenttypes of breast cancer tissue. As shown in the upper panel of FIG. 18B,the largest expression of TIGIT is observed in tumor samples with thelowest percentage of tumor cells, suggesting that TIGIT expression iscorrelated with other cells infiltrating the tumor rather than with thetumor cells themselves. The lower panel of FIG. 18A indicates that CD4⁺cells are increased in tumor samples having low percentages of tumorcells. Given the data presented herein regarding expression of TIGIT onT_(reg) and other T cell subsets, the observed high levels of TIGITexpression in the breast tumor samples with the lowest percentages oftumor cells suggests that TIGIT is being expressed by immune cell tumorinfiltrates, most likely T_(reg) infiltrates. The correlation of TIGITexpression with T cells in breast cancer samples suggests that TIGIT mayplay a role in tumor regulation. For example, a tumor may evade theimmune response of the host by recruiting/activating TIGIT⁺ T_(regs).

Example 4 Role of TIGIT in T-Cell Activation

Given the high levels of expression of TIGIT by T_(reg) and memory Tcells shown above, and the known expression of PVR on dendritic cells(Pende, D. et al., Blood 107, 2030-6 (2006)), the possibility that TIGITmight modify DC function and effect T cell activation was investigated.

(A) Function of TIGIT in Modulating T Cell Proliferation

The effect of TIGIT-Fc in a mixed lymphocyte reaction (MLR)proliferation assay was assessed using monocyte-derived human DCsmatured with TNFα. Briefly, monocytes were isolated by negativeselection of human total PBMC (Miltenyi Biotec). Immaturemonocyte-derived DC (iMDDC) were generated by incubating monocytes(3×10⁵ cells/ml) in complete RPMI 1640 medium containing 10% FBS,penicillin and streptomycin, supplemented with human recombinant IL-4(125 ng/mL, R&D Biosystems) and human recombinant GM-CSF (50 ng/mL, R&DBiosystems) in a humidified atmosphere at 37° C., 5% CO₂ for 5 days.GM-CSF and IL-4 were added again on day 2 and day 4 with fresh completeRPMI 1640 medium. After five days of culture, over 90% of the cellsexhibit an immature DC phenotype (CD14⁻, MHC class II⁺, CD80⁺, CD86⁺ andCD83^(low)) as verified by FACS analysis. These immature DC were usedhere for treatment with LPS, CD40L, TNFα, Pam3CSK4 and TSLP, and theindicated fusion proteins to induce their maturation. Phenotypicanalysis of MDDCs and cell lines was carried out by immunofluorescence.Monoclonal antibodies used for cell surface staining included PE-labeledanti-CD83, FITC-HLA-DR, PE-anti-CD86, and FITC-anti-CD80. Allincubations were performed in the presence of 10% human AB serum toprevent binding through the Fc portion of the fusions/antibodiesInhibitor studies were performed by preincubation of the indicatedmolecules with 10 μM of a MEK1 inhibitor (PD98059), 1 μg/mL anti-IL-10antibody, 10 μg/ml anti-CD32 antibody or 10 μg/ml anti-TIGIT antibodyprior to stimulation with TNFα (0.1 μg/mL). The solvent DMSO or humanIgG was used as a control. Cell culture supernatants were collectedafter 16 hours and assayed for production of IL-12 p40 by ELISA.

The effect of the blocking anti-TIGIT antibody 10A7 on T cellproliferation and activation was assessed. No effect was observed uponincubation of anti-CD3-activated CD4⁺ CD45RO⁺ T cells with 10A7. When Tcells were cultured with anti-CD3 together with autologous CD11c⁺ DC, Tcell proliferation increased two-fold (p<0.01) and IFNγ productionincreased four-fold (p<0.001) (FIG. 19C). This exacerbation of T cellactivity was observed to a lesser degree in total PBMC. In contrast,TIGIT-Fc significantly inhibited T cell activation (p<0.01) and IFNγproduction (p<0.001) in the presence of CD11c⁺ DC (FIG. 19D). When totalPBMC were activated with anti-CD3, TIGIT-Fc had a milder effect thanthat observed in the previous experiment, suggesting that the amount ofPVR present on the cells may be important for activity. No effect wasobserved on T cells alone, as expected, given that TIGIT does not bindto such cells. Anti-TIGIT antibody treatment was also found to blockT_(reg) suppression of T cell proliferation only in the presence of APC.TIGIT.Fc was further found to regulate CD11c⁺ cell function and toinhibit naïve T cell proliferation in transwell assays, indicating thatthe observed modifications in cellular behavior and proliferation weredue specifically to TIGIT binding. Taken together, these data suggestedthat TIGIT regulates T cell activation via interaction with a ligand onAPC, most likely PVR.

Both iMDDC and TNFα-matured MDDC expressed surface PVR, with the MDDCexpressing higher levels of PVR than the iMDDC (FIG. 19A). TNFα-maturedMDDC also increased proliferation of T cells over unstimulated iMDDC(FIG. 19B). In the MLR assays, the addition of TIGIT-Fc resulted in amodest yet significant decrease in proliferation, while TIGIT-Fc addedto TNFα matured MDDC reduced proliferation to baseline levels. TheTIGIT-induced inhibition of proliferation was prevented upon the furtherinclusion of anti-TIGIT antibody 10A7 or anti-PVR antibody TX21.Secreted IL-10 levels measured on day 3 were significantly higher in thecultures containing TIGIT-Fc than those containing the isotype control(45±5 pg/mL versus 29±8 pg/mL, respectively with a p=0.04). Inclusion ofanti-TIGIT antibody or anti-PVR antibody also blocked theTIGIT-Fc-induced increase in secreted IL-10 (data not shown). IFNγlevels were reduced by TIGIT-Fc treatment (data not shown). Takentogether, this data suggested that TIGIT modulates T cell activation.

To examine the effect of TIGIT⁺ T cells on TIGIT⁻ T cell proliferationin coculture, further MLR assays were performed. Briefly, CD4⁺CD45RO⁺ Tcells were isolated from human PBMC and activated for five days. On daysix, cells were restimulated with anti-CD3/anti-CD28 overnight andTIGIT⁺ cells were separately sorted from TIGIT⁻ cells by FACS. TIGIT⁻cells were CFSE labeled and mixed at a ratio of 10:1 with CD11c⁺ cellsisolated from a second donor with or without the same number of TIGIT⁺cells in culture. Culture supernatants were collected at day seven forluminex analysis of cytokine production (IFNγ or IL-17). Cellproliferation was analyzed by FACS, gating for CFSE⁺ living cells, atday eight. The results are shown in FIGS. 20A and 20B. As shown in FIG.20A, TIGIT⁺ T cells expressed lower levels of IFNγ and IL-17 than TIGIT⁻T cells. When TIGIT⁺ T cells were mixed with TIGIT⁻ T cells, theresulting culture was significantly lower in production of these twocytokines, indicating that TIGIT⁺ T cells inhibit TIGIT⁻ T cellproduction of these two cytokines. TIGIT⁺ cells also inhibitedproliferation of TIGIT⁻ T cells (FIG. 20B). This further supports theidea that TIGIT+ cells are indeed regulatory cells and can act on CD4+cells to inhibit their response either directly through secretion ofinhibitory cytokines or indirectly via engagement of PVR onantigen-presenting cells.

Based on the observation in Example 3(A) that CD4⁺CD25^(hi) T_(reg)cells in particular highly express TIGIT, assays were performed toexamine the ability of the T_(reg) T cell subset to inhibitproliferation of other immune cells. Briefly, CD4⁺CD25^(hi) T_(reg)cells were isolated from buffy coat with a MACS kit (Miltenyi) followingthe manufacturer's instructions. CD4⁺CD25⁻ cells were also prepared asthe effector T cells to be used in the assay. Antigen-presenting cell(APC) populations were isolated by standard methods, by irradiating PBMCthat had previously been depleted in T cells using MACS CD3 microbeads(Miltenyi). Isolated T_(reg), effector T cells and APC were mixedtogether at a 1:4:4 ratio and incubated with 0.5 μg/ml soluble anti-CD3.The cell mixtures were plated into wells coated with 10 μg/mL of eitheranti-TIGIT antibody 10A7 or a control IgG and cultured for four dayswith [³H]-thymidine added for the final 18 hours of incubation. Cellsfrom each well were solubilized and the amount of radioactivity in eachcell sample quantitated. The indicated percent proliferation values werecalculated relative to the amount of radioactivity observed in effectorcells in the absence of T_(reg) cells. The results are shown in FIG.21A. In wells coated with the control IgG, approximately 55% cellproliferation was observed, in keeping with the above experimentalfinding that TIGIT⁺ T cells inhibited proliferation of TIGIT⁻ T cells.Inclusion of an anti-TIGIT antibody in the wells significantly increasedthe observed proliferation, confirming that TIGIT mediates thesuppressive effect. This evidence further suggests that TIGIT⁺ T_(reg)may act as negative regulators of immune cell proliferation andfunction. In fact, when CD4⁺CD25^(hi)TIGIT⁺ T_(reg) andCD4⁺CD25^(hi)TIGIT⁻ T_(reg) were isolated and examined separately fortheir ability to suppress naïve T cell proliferation, it was found thatTIGIT⁺ T_(reg) were more potent at suppressing naïve T cellproliferation than TIGIT⁻ T_(reg). Briefly, TIGIT⁺ and TIGIT-T_(reg)were isolated by FACS. CD11c⁺ cells were positively selected usingCD11c-PE (BD Biosciences) and anti-PE microbeads (MACS). Naïve T cellswere plated on U-bottom 96 well plate at a density of 4×10⁵, along with2×10⁵ T_(reg) and 0.8×10 ⁵ CD11c⁺ antigen presenting cells. As shown inFIG. 21B, TIGIT⁺ T_(regs) were nearly twice as potent at suppressingnaïve T cell proliferation as TIGIT⁻ T_(reg) were, further supportingthe finding that TIGIT⁺ T_(reg) may act as negative regulators of immunecell proliferation and function.

(B) Knockdown of TIGIT

Using the stable cell line expressing gD-tagged TIGIT (293-TIGIT cells)constructed above, it was found that these cells did not exhibitphosphorylation of TIGIT upon interaction with exogenous PVR,cross-linked anti-TIGIT monoclonal antibody 10A7, or with pervanadatetreatment. Additionally, 10A7 treatment of these cells resulted in nosignificant effect on TCR signaling. These data suggested that eitherthe ITIM motifs in the expressed TIGIT in the constructed cells were notfunctional or that the stable cell line lacked one or more componentsnecessary for TIGIT activation.

To further elucidate cell-intrinsic functions for TIGIT, inhibitory RNA(RNAi) studies were performed. On-Targetplus gene-specific siRNAs andnegative control siRNA were obtained from Dharmacon RNAi Technology.Human CD45RO⁺ T cells were purified from buffy coat with a MACS™ kit(Miltenyi Biotec) and labeled with CFSE. siRNAs (siRNA_(control) orsiRNA_(TIGIT)) were transfected into these cells with Nucleofector™technology (Amaxxa) according to the manufacturer's instructions. After24 hours, the transfected cells were activated with plate-bound anti-CD3(5 μg/mL) alone or plus 2 μg/mL soluble anti-CD28. Some cells werecollected at day 2 or day 5 post activation for quantitative RT-PCR(qRT-PCR) or FACS analysis. T cell proliferation was determined by FACSat day 5, as described above. qRT-PCR was performed as described abovein Example 3(A), and RPL-19 mRNA levels in each sample were used asinternal controls. The TIGIT primers are given above; human CTLA4 andCD226 primers and problems were obtained from Applied Biosystems. Theprimer and probe sequences used to detect different species of murineIL-12 and IL-10 were as follows: mIL-12p40: forward primer:5′-ACATCTACCGAAGTCCAATGCA-3′ (SEQ ID NO: 55); reverse primer:5′-GGAATTGTAATAGCGATCCTGAGC-3′ (SEQ ID NO: 56); probe:5′-TGCACGCAGACATTCCCGCCT-3′ (SEQ ID NO: 57); mIL-12p35: forward primer:5′-TCTGAATCATAATGGCGAGACT-3′ (SEQ ID NO: 58); reverse primer:5′-TCACTCTGTAAGGGTCTGCTTCT-3′ (SEQ ID NO: 59); probe:5′-TGCGCCAGAAACCTCCTGTGG-3′ (SEQ ID NO: 60); mIL-10: forward primer:5′-TGAGTTCAGAGCTCCTAAGAGAGT-3′ (SEQ ID NO: 61); reverse primer:5′-AAAGGATCTCCCTGGTTTCTC-3′ (SEQ ID NO: 62); probe:5′-TCCCAAGACCCATGAGTTTCTTCACA-3′ (SEQ ID NO: 63).

RNAi specific for TIGIT were employed to specifically knock down TIGITexpression in primary human CD45RO⁺ T cells, which normally express highlevels of TIGIT (FIGS. 10A-1 to 10A-2). The efficacy of TIGIT knockdownusing this method was assessed by qRT-PCR and FACS analysis (FIGS. 28A,28B, and Table 7). By the second day of treatment, TIGIT transcriptionwas reduced by >90% by the siRNA_(TIGIT) treatment as compared to ascrambled siRNA_(control), while CTLA4 mRNA (a control protein) wasunchanged by the treatment. The reduction in TIGIT mRNA resulted in adecrease of cell surface TIGIT from an average of 25% to <2% of the Tcells (FIG. 28B). By day 5, TIGIT expression in those same cells was 70%reduced as compared to expression in control cells (FIGS. 28A and 28B,and Table 7). Knockdown of TIGIT had no significant effect on T cellproliferation in response to anti-CD3 (either at suboptimal or optimalconcentrations) or to anti-CD3 plus anti-CD28 (FIG. 28C). Similarly,knockdown of TIGIT also had no observed effect on production of thecytokines IL-2, IL-4, IL-10, or IFN-γ (FIG. 28E). Furthermore, treatmentof the cells with anti-TIGIT antibody 10A7 had no observed effect onactivation of T cells expressing TIGIT under the same conditions asdescribed above (FIG. 28D).

TABLE 7 TIGIT RNAi Knockdown Efficiency siRNA_(control) siRNA_(TIGIT)C_(T)* Day 2 Day 5 Day 2 Day 5 TIGIT mRNA 23.3 ± 0.1 24.0 ± 0.0 31.2 ±0.6 29.8 ± 0.1 CTLA4 mRNA 27.2 ± 0.4 24.3 ± 0.3 27.6 ± 0.3 23.8 ± 0.3*C_(T) values are given as the C_(T) value ± standard deviation forTIGIT or CTLA4

Example 5 Effect of TIGIT on Cytokine Production

To determine whether TIGIT had a direct effect on DCs other than theabove-described general effect on T cell maturation, DC maturation andfunction in the presence and absence of TIGIT-Fc was assessed. Theresult in Example 4 regarding TIGIT's ability to modulate IFNγ and IL-17production in mixed T cell populations suggested that further studies ofcytokine production by DC treated with TIGIT-Fc should be performed.Untreated T cells were purified by negative selection (CD4 T cellisolation kit, Miltenyi Biotech) to a purity of >95%. Cells wereresuspended in complete RPMI 1640 medium with standard nutritionalsupplements. Allogenic T cells (2×10⁵) were cultured in the absence(medium alone) or presence of iMDDCs and MDDCs at the indicated ratio in96-well U-bottomed μplates (Nunc) in 200 μA of medium per well. Cellswere cultured for 72 hours followed by an 18 hour pulse with 1 μCi(0.037 MBq) of [³H]thymidine (Amersham). Cells were transferred to aUnifilter-96 plate GF/C using a cell harvester and [³H]thymidineincorporation was measured in scintillation fluid using a scintillationcounter (Canberra Packard Ltd.). All determinations were carried out intriplicate. Cytokine production by iMDDCs was analyzed on supernatantscollected on day 5 of culture and stored at −80° C. The same MDDCs werematured in the presence or absence of indicated stimuli for 24 hours inthe presence or absence of TIGIT-Fc or TIGIT-Fc-DANA. After 48 hours ofstimulation, supernatants were collected and stored at −80° C. Cytokineconcentrations were measured by ELISA (R&D Biosystems) according to themanufacturer's instructions, or by using LINCOplex antibody-immobilizedbeads (LINCO Research) with detection by a Luminex 100 instrument(Luminex) according to the manufacturer's instructions.

When TIGIT-Fc, TIGIT-Fc-DANA, or CD226-Fc were added to iMDDC duringmaturation with TNFα or soluble CD40L, IL-12/23p40 production andIL-12p70 production were significantly reduced as compared to treatmentwith isotype-matched control (p=0.007 and p=0.03, respectively), tolevels comparable for iMDDC (FIGS. 22A-1 to 22A-3). Conversely, secretedIL-10 was increased by TIGIT-Fc, TIGIT-Fc-DANA, or CD226-Fc treatmentrelative to treatment with isotype-matched control (p=0.027 and p=0.18,respectively) (FIGS. 22A-1 to 22A-3). TGFβ secretion was also increasedin iMDDC in response to TIGIT-Fc treatment (see FIG. 22D). TIGIT-Fc didnot, however, affect the ability of iMDDC to mature to MDDC, since CD80,CD86, CD83 and HLA-DR were equivalently upregulated in isotype controlcultures (FIG. 22B). Notably, the TIGIT-PVR interaction did not directlyinduce DC maturation.

The effect of TIGIT-Fc, TIGIT-Fc-DANA and CD226-Fc on TLR-mediated DCmaturation pathways was also examined. Treatment with each of the threeFc proteins exhibited similar, though less robust increase of IL-10production from LPS (TLR4-matured MDDC (p<0.01), a decrease inIL-12/23p40 (p=0.07 to 0.18) and significant decrease in IL-12p70production (p<0.05 for all fusion proteins) (see FIGS. 22A-1 to 22A-3),and had no effect on the TLR2 maturation pathway. This modulation ofIL-10 and IL-12p40 production by TIGIT treatment of DC was similarwhether TIGIT-Fc was added to monocytes during differentiation withGM-CSF and IL-4, or when only added during the maturation phase (datanot shown). The effects of TIGIT-Fc on IL-10 and IL-12p40 production iniMDDC not undergoing maturation were modest, but since the observedlevels of those cytokines in iMDDC were low, statistically significanteffects may have been difficult to detect (FIG. 22B). Notably, CD226functioned similarly to TIGIT in these assays, supporting a role for PVRin MDDC. Given that CD226 has an ITAM motif and may act to enhance TCRsignals (Dalhardon et al. J. Immunol. 175: 1558-1565 (2005)), the degreeof expression of TIGIT and/or CD226 on different subsets of T cells maycontribute to differential regulation of local inflammatory responses invivo (FIG. 10, FIG. 29).

The levels of production of other proinflammatory cytokines by DCtreated with TIGIT-Fc were also determined. Both IL-6 and IL-18production was significantly reduced by TIGIT-Fc treatment in allmatured MDDC populations. IL-12p40 is a known subunit of both IL-12p70and IL-23, so the levels of production of both of those cytokines weremeasured in TIGIT-Fc-treated MDDC cultures. Compared to controlcultures, TIGIT-Fc treatment resulted in significantly decreasedIL-12p70 production by MDDC matured with TNFα or CD40L (FIG. 22C). IL-23levels were relatively low and barely detectable under the assayconditions. TIGIT-Fc reduced both IL-6 and IL-18 under all matured MDDCconditions, but due to donor variability the observed reduction was notstatistically significant.

To assess whether the observed effect of TIGIT-Fc required cross-linkingof PVR, an Fc-mutated version of TIGIT-Fc in which FcγR binding wascompletely abrogated (TIGIT-Fc-DANA, described in Example 1) was used.As shown in FIGS. 22A-1 to 22A-3, both TIGIT-Fc and TIGIT-Fc-DANAequally and significantly inhibited IL-12p40 and enhanced IL-10production from DC matured with TNFα. This result indicated thatcytokine skewing by the TIGIT fusion protein was not dependent onFc-mediated cross-linking

The ability of TIGIT to modify the cytokine production pattern from DCwas not observed under all in vitro maturation conditions. The effectwas most pronounced on TNFα, soluble CD40L and LPS (TLR4)-inducedmaturation pathways, whereas TLR2-mediated maturation remainedunaffected. It has been shown that LPS and Pam3CSK4 activate ERK and p38to various extents: LPS mainly activates p38 and Pam3CSK4 treatmentresults in high ERK kinase activity. Thus it is not surprising thatTIGIT-Fc treatment of Pam3CSK4-matured DC showed little effect (seeFIGS. 22A-1 to 22A-3). The differential ability of these and otherstimuli such as TNFα and CD40L to regulate the ERK/p38 pathways issignificant in determining the outcome of MDDC function. Not only haveDC been demonstrated to expand Tregs, but DC can also break T_(reg)tolerance and induce activation and IL-2 production (Fehervari, Z. &Sakaguchi, S., Curr Opin Immunol 16, 203-8 (2004)). The ability of TIGITto modify DC under some maturation conditions but not others suggeststhat TIGIT modulation is one method by which T_(reg) and activated Tcells may fine-tune DC function.

Studies of TIGIT function were also performed in a mouse model lacking Band T cells but which have macrophages and dendritic cells (scid mice).Briefly, CB17/SCID mice (6-8 weeks old) were treated once intravenouslywith 200 μg of TIGIT.Fc, TIGIT.DANA, or a control anti-ragweed antibody.Anti-CD40 monoclonal antibody or isotype control (200 μg/mice) wasadministered six hours later. Serum was collected 16 hours later toanalyze levels of IL-10, MCP-1, IL-12p40 and IL-12p70 by ELISA assay.Administration of TIGIT-Fc or TIGIT-Fc-DANA in scid mice stimulatedIL-10, and IL-12p40 production and decreased IL-12p70 production (FIGS.13A-C). This finding was consistent with the in vitro data above, andsuggests that TIGIT does not require B or T cells to exert its cytokinemodulatory effects.

From the preceding examples, expression of TIGIT was restricted to Tcells and NK cells, with the highest expression found in T_(regs).CD226, the low affinity ligand for PVR, is not expressed on T_(regs)despite expression on activated T cells (Abbas, A. R. et al., GenesImmun 6, 319-31 (2005); Dardalhon, V. et al., J Immunol 175, 1558-65(2005)). Although the balance of TIGIT and CD226 in vivo remains to bedetermined, the higher affinity of TIGIT for PVR suggests it plays adominant role when both are co-expressed. Taken together, the highexpression of TIGIT on activated T cells and T_(reg) and interaction ofTIGIT with PVR to induce IL-10 and to inhibit proinflammatory cytokinerelease from mature DC suggest that TIGIT provides a feedback mechanismto down-regulate immune response.

Example 6 Effect of TIGIT on PVR Signaling

Since the MAPK signaling pathway is important in regulating the IL-10pathway (Xia, C. Q. & Kao, K. J., Scand J Immunol 58, 23-32 (2003)), theactivity of several members of the MAPK pathway was assessed inTIGIT-treated MDDC. CHO-PVR were serum-starved for three hours thentreated with 50 μg/mL TIGIT-Fc or not treated for 15 minutes at 37° C.Cells were homogenized and membrane proteins were extracted using aPlasma Membrane Extraction Kit (BioVision) and subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) undernon-reducing conditions, followed by transfer to nitrocellulosemembranes (BioRad). Membranes were blocked with 5% BSA in 50 mM Tris-HCl(pH 7.6), 150 mM NaCl, 0.1% Tween-20, and then probed withanti-phosphotyrosine-HRP (BD Bioscience), stripped with Restore Buffer(Pierce), and re-probed with anti-PVR goat polyclonal antibody (R&DSystems). Day 5 iMDDC were treated with 10 μg/mL TIGIT-Fc or controlhuman IgG for the indicated time period at 37° C. Total cell lysateswere prepared in RIPA buffer and subjected to SDS-PAGE under reducingconditions and transferred to Immobilon polyvinylidene difluoridemembrane (PVDF, Millipore). After blocking with 1% BSA in 50 mM Tris-HClpH 7.6, 150 mM NaCl, 0.1% Tween-20, followed by chemiluminescent proteindetection. For reprobing, membranes were incubated in stripping buffer(62.5 mM Tris-HCl pH 6.7, 100 mM (3-mercaptoethanol, 2% SDS) for 30minutes at 50° C. with occasional agitation. Detection ofphosphotyrosine, phosphor-p38MAPK, and phosphor-ERK was carried outusing polyclonal antibodies specific for anti-phosphotyrosine (Upstate),anti-phospho-p38MAPK (Cell Signaling Technology), and a monoclonalanti-phospho-p44/42 MAPK (Cell Signaling Technology). As a control forprotein loading, blots were re-probed with polyclonal antisera againstERK (Cell Signaling Technology), p38MAPK (Cell Signaling Technology) orβ-actin (NeoMarkers), β-catenin (BD Pharmingen) or active β-catenin(Upstate).

The data shown in FIG. 23A demonstrate that PVR is phosphorylated uponbinding to TIGIT (compare faint phosphorylated tyrosine band observed inisotype-matched control versus TIGIT-treated cells, while overallamounts of PVR remained constant as indicated by the equivalently darkbands in the lower portion of the figure). This suggested that TIGITbinding initiates a signaling function mediated by PVR. Increasedphosphorylation of pERK dimer (91 KD) but not monomer (42 KD) wasobserved in TIGIT-Fc and TIGIT-Fc-DANA-treated iMDDC (FIG. 23B). Incontrast, p38 activity was not affected (FIG. 23B). A recent reportsuggested that stimulation of E-cadherin and induction of activeβ-catenin caused murine bone marrow-derived DC to mature intotolerogenic DC capable of inhibiting immune responses in vivo (Jiang, A.et al., Immunity 27, 610-24 (2007)). Here, when human MDDC were treatedwith TIGIT-Fc the active form of the β-catenin pathway was induced, aneffect not observed with the isotype matched control (FIG. 23C).

These results suggested that TIGIT, through its interaction with PVR,modulates ERK activity and thus cytokine production by MDDC. To confirmthis observation, an ERK kinase specific inhibitor was added togetherwith TIGIT-Fc to MDDC cultures, and the levels of secreted IL-12 fromthose cultures were determined. TIGIT-Fc-mediated down-regulation ofIL-12p40 production was reversed in the presence of the ERK inhibitor(FIG. 24A). A similar effect was observed when a neutralizing anti-IL-10antibody was included in the culture (see FIG. 24B). TIGIT-modulatedcytokine production from MDDC was also blocked by anti-TIGIT antibody10A7 or a blocking anti-PVR antibody (FIG. 24B). Together, these resultsindicated that TIGIT-PVR ligation affects ERK kinase activity andincreases the ratio of IL-10/IL-12 cytokine production in DC relative toother produced cytokines

Example 7 Impact of TIGIT-Modulated MDDC on T-Cell Activation

To determine if the effect of TIGIT on DC cytokine production hadfunctional consequences, experiments were performed to assess the effectof TIGIT modulation of MDDC on T cell proliferation and cytokineproduction. TIGIT-Fc-treated MDDC (matured with either TNFα or sCD40L)were cultured with T cells in an MLR response as described above, andthe effect on T cells was monitored. T cell proliferation was inhibitedby an average of 50% (p<0.05) when cultures containing TIGIT-modified DCwere compared with control DC (FIG. 25A). Additionally, IL-2 levels inthe cultures were two-fold reduced (p<0.01) (FIG. 25B)). This datacorrelates with the decrease in IL-12 and increase in IL-10 productionin DC treated with TIGIT, as described in the preceding examples.Overall, TIGIT-modified MDDC inhibited T cells, which suggests thatTIGIT can regulate DC functional capabilities once DC are fully matured.Notably, addition of TIGIT-Fc to MDDC-T cell cultures inhibitedproliferation of the T cells, which indicates that TIGIT-Fc does notneed to be present at the initiation of DC maturation to modify the DC.

The impact of TIGIT treatment on the expression of other cell-surfacemolecules in activated human MDDC was also investigated. It had beenknown that the expression of certain immunoglobulin-like transcripts(ILT) receptors on DC is modulated in response to activation of thosecells (Velten et al., Eur. J. Immunol. 34: 2800-2811 (2004); Ju et al.,Gene 331: 159-164 (2004)). For example, expression of the ILT2 and ILT3receptors is down-regulated in CpG-DNA-activated DC, and expression ofILT2, ILT3, ILT4, and ILT5 is up-regulated in IL-10-induced DC. Giventhat TIGIT stimulates IL-10 production in DC, the impact of TIGIT on ILTexpression in activated DC was examined. iMDDC were isolated asdescribed above. Certain populations of iMDDC were activated with TNF orCD40L, and also treated with TIGIT-Fc or an isotype-matched control.Treated cells were sorted by FACS based on their expression ofimmunoglobulin-like transcript 2, 3, or 5 (ILT2, ILT3, or ILT5). Asshown in FIG. 26, activation of iMDDC downregulates ILT2, ILT3, and ILT5expression. In contrast, activation and simultaneous treatment withTIGIT-Fc results in a decreased down-regulation of ILT2, ILT3, and ILT5expression relative to the down-regulation seen in iMDDC activated butuntreated with TIGIT-Fc. This observed effect may be due to the abilityof TIGIT to stimulate IL-10 production in DC; IL-10-expressing DC areknown to be tolerogenic and to express higher levels of ILTs. However,down-regulation of ILTs such as ILT2, 3, and 5 may also be a directeffect of TIGIT, and provide another method by which TIGIT inducestolerance.

To determine whether the observed in vitro effects of TIGIT treatment onT cell activation could be translated to an in vivo situation, theeffects of TIGIT-Fc treatment were compared to those of CTLA4-Fc, awell-documented inhibitor of T cell response (Linsley, P. S. et al.,Science 257, 792-5 (1992)) in a delayed-type hypersensitivity (DTH)response. Briefly, 8-10 week old C57BL/6 mice were immunizedsubcutaneously in the base of the tail with 100 μg keyhole limpethemocyanin (KLH) (Sigma) in 100 μL CFA (Difco Laboratories). One cohortof animals (n=10) was treated on days 1, 4 and 6 with 100 μg of murineTIGIT-Fc, TIGIT-Fc-DANA, CTLA-4-Fc or negative isotype controlanti-ragweed IgG2a by intraperitoneal injection. On day 6, right andleft ear thickness was measured. The right ear was then injected with 25μL saline and the left ear was challenged with 30 μg KLH in 25 μLsaline. On day 7, right and left ear thicknesses were again measured,and the difference between day 7 and day 6 ear thicknesses was definedas ear swelling. Ear swelling in ears injected with saline alone wasless than 0.02 mm for each treatment group. After ear swellingmeasurement, mice were euthanized and spleens harvested. Single-cellsuspensions were prepared and cultured in 96-well flat-bottom plates ata density of 1×10⁶ cells/ml (200 μL/well) in DMEM containing 10% FBS, 2mM glutamine, penicillin (100 U/ml) and streptomycin (100 μg/mL). Cellswere cultured in medium alone or in the presence of variousconcentrations of KLH. As a positive control for T cell activation,cells were cultured on wells precoated with 5 μg/mL anti-CD3 (BDBiosciences) with 2 μg/mL soluble anti-CD28 (BD Biosciences). Forproliferation analysis, 1 μCi [³H]thymidine (Perkin Elmer) was added toeach well in a volume of 50 μL for the last 18 hours of a four-dayculture, cells were harvested and incorporation of [³H]thymidine wasmeasured by liquid scintillation counting.

Significantly lower ear swelling was measured in TIGIT-Fc andCTLA4-Fc-treated mice as compared to the control treatment, and potencywas similar for both treatment groups (p<0.0001 for both groups) (FIG.27A). There was no statistical difference between TIGIT-Fc and CTLA4-Fc(p=0.07). Significantly, in IL-10 deficient mice, TIGIT-Fc had no effecton DTH responses, in spite of inhibition of DTH with CTLA4-Fc (p=0.004),supporting the role of IL-10 in TIGIT-PVR function. TIGIT-Fc-DANA wassimilar in its effects at inhibition of DTH as TIGIT-Fc, demonstratingthat TIGIT-Fc did not require Fc-mediated cross-linking of PVR.Anti-TIGIT had no effect on DTH (FIG. 27C). Assays were performed todetermine in vitro recall responses to KLH in treated mice anddemonstrated that proliferation, IL-2 and IFNγ cytokine production wassignificantly decreased in TIGIT-Fc-treated wild type but notIL-10-deficient mice (FIGS. 27D-G).

CD11c⁺ DC were isolated from spleens in the DTH mice at studytermination (day 7) and the effect of TIGIT-Fc on DC proliferation andcytokine profiles was assessed by qRT-PCR, as described above. Splenic Tcells isolated from TIGIT-Fc and CTLA4-Fc-treated animals did notproliferate in response to KLH in recall assays as compared toisotype-treated control animals (p<0.001 for both treatment groups)(FIG. 27B). This result indicates that TIGIT may be important duringboth T-cell priming and the effector phase of T-cell driven immuneresponses. Similar to the in vitro data obtained above from the MDDCstudies, CD11c⁺ cells isolated from TIGIT-Fc-treated mice had increasedIL-10 mRNA (p<0.05) and decreased IL-12/23p40 and IL-12p35 mRNA,although these latter measurements did not reach statisticalsignificance (p=0.07 and 0.08, respectively) (FIG. 27H). However, theTIGIT-Fc treatment had only a minor effect on IL-12p40/p35 transcriptionin CD11c⁺ cells derived from IL-10 KO mice, indicating thatTIGIT-mediated down-regulation of IL-12p40/p35 mRNA levels is specificand TIGIT-mediated upregulation of IL-10 is required for down-regulationof proinflammatory cytokine IL-12 in this model.

Example 8 TIGIT Deficient Mice

TIGIT knockout mice were generated using standard techniques. To confirmthe absence of a functional TIGIT gene in these mice, total T cells wereisolated from spleens of knockout or wild-type mice, and subsequentlyincubated with anti-CD3 antibodies and anti-CD28 antibodies for threedays. Total RNA was isolated from the cells using an RNeasy kit (Qiagen)and subjected to real-time RT-PCR to measure TIGIT mRNA. CD96 mRNAlevels were also assessed as a control. The results of the studydemonstrated that the knockout mice were deficient in TIGIT expression.

Immune cell populations from mesenteric lymph nodes were examined in 9month-old TIGIT knockout mice in comparison with wild-type mice, usingFACS analyses as described in Example 3A. The TIGIT knockout micedisplayed increased numbers of memory CD4⁺ T cells, mDC, pDC, monocytes,CD11c⁺ PVR^(hi) T cells, and overall B cells as compared to wildtypemice. The populations of naïve and mature CD4⁺ cells were similarbetween the knockout and wildtype mice. The knockout mice were alsofound to have increased numbers of MZB (B220⁺CD21^(hi)), NKT (DX5⁺CD4⁺or DX5⁺CD8⁺), and memory CD8⁺ T cells in spleen, relative to wildtypemice. This increased level of memory CD8⁺ T cells was also observed inmesenteric lymph nodes and Peyer's patch cells in the knockout mice. Theincrease in pDC and monocyte cell numbers observed in the mesentericlymph node of the knockout mice was also observed in spleen and Peyer'spatches of those mice, though the difference in levels relative to thosein wildtype mice was less pronounced than in the mesenteric lymph node.The activity of T cells isolated from the TIGIT deficient mice was alsoinvestigated.

Briefly, total splenocytes were isolated from 9-month-oldTIGIT-deficient mice and wild-type littermates. 10⁶ cells from each typeof mice were seeded onto flat-bottom 96-well plates and stimulated withplate-bound anti-CD3 (10 μg/mL) plus anti-CD28 (2 μg/mL). On the secondday, supernatants were collected and cytokine production was analyzed byLuminex. Cells were collected and subjected to FACS, sorting by thepresence of intracellular IFNγ and IL-4. Cell proliferation was measuredby ³H-thymidine incorporation, as described in Example 3A. MLR assayswere performed generally according to the methods described in Example4A. Specifically, CD4⁺ T cells were isolated from spleens ofTIGIT-deficient mice or wild-type littermates by negative isolation(MACS). T-cell-depleted Balb/C splenocytes were irradiated at 3000 radand used as antigen presenting cells. 2×10⁵ CD4⁺ T cells were stimulatedwith 1 μg/mL soluble anti-CD3 (T cells only) or mixed with allogenicantigen presenting cells at a 1:2 ratio. Proliferation was measured onthe third day by ³H-thymidine incorporation, as described in Example 3A.In a second experiment, the MLR assay was performed identically, but theCD4⁺ T cells were isolated from Balb/c mice and the antigen presentingcells were prepared from TIGIT-deficient mice or from wild-type mice.

The TIGIT-deficient mouse T cells proliferated similarly to T cells fromwild-type mice in a standard proliferation assay (FIG. 30A, left panel).However, in the presence of antigen presenting cells, TIGIT-deficient Tcells had increased proliferation relative to wild-type T cells (FIG.30A, middle panel). Notably, antigen-presenting cells fromTIGIT-deficient mouse spleen stimulated proliferation of wild-type Tcells to the same extent as antigen presenting cells taken fromwild-type mice (FIG. 30A, right panel). Combined, this data suggeststhat T cells are downregulated in proliferation by a mechanism involvingTIGIT expressed on those T cells, rather than on antigen-presentingcells, and further confirms the activity of TIGIT in the down-regulationof T cell response. A greater proportion of the TIGIT-deficient mouse Tcells had high intracellular IFNγ levels than the wild-type mouse Tcells (FIG. 30B). Cytokine production analyses of supernates fromTIGIT-deficient and wild-type T cells showed that IFNγ and TNFαproduction/secretion was increased in the TIGIT-deficient T cellsrelative to the wild-type T cells, while IL-2, IL-4, IL-5, IL-10, andIL-12p70 levels remained consistent between the two cell populations.

1. An isolated polypeptide comprising an amino acid sequence comprisingone or more of the following amino acids: an alanine at an amino acidposition corresponding to amino acid position 67 of human TIGIT, aglycine at an amino acid position corresponding to amino acid position74 of human TIGIT, a proline at an amino acid position corresponding toamino acid position 114 of human TIGIT, and a glycine at an amino acidposition corresponding to amino acid position 116 of human TIGIT.
 2. Thepolypeptide of claim 1, wherein the polypeptide is not PVR, PVRL 1,PVRL2, PVRL3, PVRL4, TIGIT, CD96, or CD226.
 3. The polypeptide of claim1, wherein the polypeptide further comprises one or more of: an aminoacid selected from valine, isoleucine, and leucine at an amino acidposition corresponding to amino acid position 54 of human TIGIT, anamino acid selected from serine and threonine at an amino acid positioncorresponding to amino acid position 55 of human TIGIT, a glutamine atan amino acid position corresponding to amino acid position 56 of humanTIGIT, a threonine at an amino acid position corresponding to amino acidposition 112 of human TIGIT, and an amino acid selected fromphenylalanine and tyrosine at an amino acid position corresponding toamino acid position 113 of human TIGIT.
 4. The polypeptide of claim 1,wherein the polypeptide further comprises one or more structuralsubmotifs selected from the following: a. an amino acid selected fromvaline and isoleucine at amino acid position 54-an amino acid selectedfrom serine and threonine at amino acid position 55-a glutamine at aminoacid position 56; b. an alanine at position 67-any amino acid at each ofamino acid positions 68-73-a glycine at amino acid position 74; and c. athreonine at amino acid position 112-an amino acid selected fromphenylalanine and tyrosine at amino acid position 113-a proline at aminoacid position 114-any amino acid at amino acid position 115-a glycine atamino acid position 116, and wherein the numbering of the amino acidpositions corresponds to the amino acid positions of human TIGIT.
 5. Amethod of determining whether a test polypeptide is a member of the TLPfamily of polypeptides comprising aligning the amino acid sequence ofthe test polypeptide with an amino acid sequence of one or more membersof the TLP family of polypeptides and assessing the presence or absencein the test polypeptide amino acid sequence of any of the amino acids asset forth in claim
 1. 6. A method for identifying one or more members ofthe TLP protein family by identifying proteins in one or more sequencedatabases whose amino acid sequences comprise at least one of the aminoacids as set forth in claim
 1. 7. An isolated agent that specificallyinteracts with one or more conserved or substantially conserved regionsof the TLP family members of claim
 1. 8. The agent of claim 7, whereinthe agent is an antagonist of the expression and/or activity of a TLPfamily member.
 9. The agent of claim 8, wherein the antagonist isselected from a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.
 10. The agent of claim 7, whereinthe agent is an agonist of the expression and/or activity of a TLPfamily member.
 11. The agent of claim 10, wherein the agent is selectedfrom an agonizing antibody or antigen-binding fragment thereof, anagonizing peptide, and a small molecule or protein that activates TIGITbinding to PVR and/or TIGIT intracellular signaling mediated by PVR. 12.A method of identifying or detecting one or more TLP family members bycontacting a putative TLP family member polypeptide with the agent ofclaim 7 and determining the binding of the agent to the putative TLPfamily member.
 13. A method of determining whether a test immune cell isan activated or normal T_(reg), memory T cell, NK cell, or T_(Fh) cell,comprising assessing the level of expression of TIGIT in the test immunecell and comparing it to the level of expression of TIGIT in a knownactivated or normal T_(reg), memory T cell, NK cell, or T_(Fh) cell, orby comparing the level of expression of TIGIT in the test immune cell toknown standard TIGIT expression value(s).
 14. A method for modulatingimmune system function and/or activity comprising modulating the bindingof TIGIT to one or more of PVR, PVRL3, and PVRL2.
 15. An anti-TIGITantibody or a fragment thereof comprising at least one HVR comprising anamino acid sequence selected from the amino acid sequences set forth inSEQ ID NOs: 23-28 or SEQ ID NOs: 31-36.
 16. The anti-TIGIT antibody orantigen-binding fragment thereof of claim 15, wherein the antibody lightchain comprises the amino acid sequence set forth in SEQ ID NOs: 21 or29.
 17. The anti-TIGIT antibody or antigen-binding fragment thereof ofclaim 15, wherein the antibody heavy chain comprises the amino acidsequence set forth in SEQ ID NOs: 22 or
 30. 18. The anti-TIGIT antibodyor antigen-binding fragment thereof of claim 15, wherein the antibodylight chain comprises the amino acid sequence set forth in SEQ ID NOs:21 or 29 and the antibody heavy chain comprises the amino acid sequenceset forth in SEQ ID NOs: 22 or
 30. 19. The anti-TIGIT antibody orantigen-binding fragment thereof of claim 15, wherein the antibody isselected from a humanized antibody, a chimeric antibody, a bispecificantibody, a heteroconjugate antibody, and an immunotoxin.
 20. Theanti-TIGIT antibody or antigen-binding fragment thereof of claim 15,wherein the at least one HVR is at least 90% identical to an HVR setforth in any of SEQ ID NOs: 23-28 or 31-36.
 21. The anti-TIGIT antibodyor fragment thereof of claim 15, wherein the light chain and/or heavychain comprise amino acid sequences at least 90% identical to the aminoacid sequences set forth in SEQ ID NOs: 21 or 29, or 22 or 30,respectively.
 22. A method of modulating a CD226-PVR interaction and/ora CD96-PVR interaction comprising administering at least one of TIGIT,an agonist of TIGIT expression and/or activity, or an antagonist ofTIGIT expression and/or activity in vivo or in vitro.
 23. The method ofclaim 22, wherein TIGIT or an agonist of TIGIT expression and/oractivity is administered and the CD226-PVR interaction and/or theCD96-PVR interaction is inhibited.
 24. The method of claim 22, whereinan antagonist of TIGIT expression and/or activity is administered andthe CD226-PVR interaction and/or the CD96-PVR interaction is stimulated.25. A method of modulating immune cell function and/or activity bymodulating TIGIT and/or PVR expression and/or activity, or by modulatingthe intracellular signaling mediated by TIGIT binding to PVR.
 26. Themethod of claim 25, wherein the modulating is decreasing or inhibitingproliferation of one or more immune cells or proinflammatory cytokinerelease by one or more immune cells by treating the cells in vitro or invivo with TIGIT, an agonist of TIGIT expression and/or activity, anagonist of PVR expression and/or activity, or by stimulatingintracellular signaling mediated by TIGIT binding to PVR.
 27. The methodof claim 25, wherein the modulating is increasing or stimulatingproliferation of one or more immune cells or proinflammatory cytokinerelease by one or more immune cells by treating the cells in vitro or invivo with an antagonist of TIGIT expression and/or activity, anantagonist of PVR expression and/or activity, or by inhibitingintracellular signaling mediated by TIGIT binding to PVR.
 28. A methodof inhibiting an immune response by administering in vitro or in vivoTIGIT, an agonist of TIGIT expression and/or activity, an agonist of PVRexpression and/or activity, or by stimulating intracellular signalingmediated by TIGIT binding to PVR.
 29. A method of increasing orstimulating an immune response by administering in vitro or in vivo anantagonist of TIGIT expression and/or activity, an antagonist of PVRexpression and/or activity, or by inhibiting intracellular signalingmediated by TIGIT binding to PVR.
 30. A method of modulating the typeand/or amount of cytokine production from an immune cell by modulatingTIGIT or PVR expression and/or activity in vitro or in vivo.
 31. Themethod of claim 30, wherein proinflammatory cytokine production isstimulated and/or increased by administration of an antagonist of TIGITexpression and/or activity, an antagonist of PVR expression and/oractivity, or by inhibiting intracellular signaling mediated by TIGITbinding to PVR.
 32. The method of claim 30, wherein proinflammatorycytokine production is inhibited by administration of an agonist ofTIGIT expression and/or activity, an agonist of PVR expression and/oractivity, or by stimulating intracellular signaling mediated by TIGITbinding to PVR.
 33. A method of stimulating ERK phosphorylation and/orintracellular signaling through the ERK pathway in one or more immunecells comprising treating the one or more immune cells with TIGIT, anagonist of TIGIT expression and/or activity, or an agonist of PVRexpression and/or activity.
 34. A method of diagnosing or assessing theseverity of an immune-related disease relating to aberrant immune cellresponse in a subject comprising assessing the expression and/oractivity of TIGIT in a sample from the subject and comparing theexpression and/or activity of TIGIT to a reference amount of TIGITexpression and/or activity or the amount of TIGIT expression and/oractivity in a sample from a normal subject.
 35. (canceled)
 36. A methodof preventing, treating and/or lessening the severity of animmune-related disease relating to aberrant immune cell response in asubject comprising modulating the expression and/or activity of TIGIT inthe subject.
 37. (canceled)
 38. The method of claim 36, wherein theimmune-related disease is selected from psoriasis, arthritis,inflammatory bowel disease or cancer.